Screening assay to identify modulators of the sleep/wake cycle

ABSTRACT

Screening assays for identifying agents that modulate BK channel activity and further modulate the sleep/wake cycle in a subject, circadian regulated locomotor activity in a subject, or both are provided, as are agents identified using such screening assays. Also provided are methods of modulating the sleep/wake cycle in a subject and methods of modulating circadian regulated locomotor activity in a subject by administering an agent that modulates BK channel activity to the subject, for example, an agent identified by a screening assay as disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 10/464,817 filed Jun. 17, 2003, now issued as U.S. Pat. No.7,427,489; which claims the benefit under 35 USC § 119(e) to U.S.Application Ser. No. 60/389,759 filed Jun. 17, 2002, now abandoned. Thedisclosure of each of the prior applications is considered part of andis incorporated by reference in the disclosure of this application.

GRANT INFORMATION

This invention was made in part with government support under Grant No.NH51573 awarded by the National Institutes of Health. The government hascertain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the circadian regulation ofthe sleep/wake cycle, and more specifically to methods of identifyingagents that affect calcium activated potassium channel activity andthereby modulate the sleep/wake cycle, and to methods of modulating thesleep/wake cycle by affecting calcium activated potassium channelactivity.

2. Background Information

Most organisms undergo a rhythmic pattern of sleep and wakefulness thatcycles over a twenty-four hour period and generally is linked to theday/night cycle. In human adults, for example, sleep onset generallybegins about four to five hours after nightfall, and spontaneousawakening occurs about one to two hours after sunrise (see Young andKay, Nat. Rev. Genet. 2:702, 2001). Many individuals, however, sufferfrom abnormalities in the normal sleep/wake cycle, including, forexamples, individuals that suffer from insomnia and individuals thatsuffer from disorders such as narcolepsy, in which sleep onset can occurat any time of day, or familial advanced sleep phase syndrome (FASPS),in which the time of sleep onset and awakening occur earlier than normal(see, for example, Young and Kay, supra, 2001).

A genetic mutation was recently linked to FASPS in humans, and is thefirst example of a genetic defect associated with a defect in thesleep/wake cycle in humans (Toh et al., Science 291:1040, 2001). Theidentification of such a mutated gene, and its normal counterpart,provide a means to develop assays for identifying drugs that correct, orat least decrease, the effect due to the mutation, thus allowing anindividual with the mutation to have a more normal pattern of sleep andwakefulness. Although drugs that ameliorate the effect due to themutation in FASPS will be a great help to individuals having thatmutation, the drugs are not likely to be useful for individualssuffering from other sleep disorders such as insomnia. As such, themajority of individuals suffering from disorders of the sleep/wake cyclesuch as insomnia must continue to rely on relatively non-specific drugs,including prescription drugs such as benzodiazepine agonists andover-the-counter drugs, which often contain antihistamines. Such drugs,however, act generally and, while they can assist in helping a personsleep, they also can have undesirable side effects, including causingconfusion and loss of balance.

Clearly, it would be preferable to identify additional specific genesinvolved in regulating the sleep/wake cycle so that individual sufferingfrom sleep disorders so that the gene products could be used as targetsin screening assays to identify drugs useful for specifically modulatingthe sleep/wake cycle. Microarray technology provides a means to identifythe expression of a large number of genes in a single assay and,therefore, provides a powerful tool for identifying genes of interest,including those involved in regulating the sleep/wake cycle. Amicroarray is formed by linking a large number of discretepolynucleotide sequences, for example, a population of polynucleotidesrepresentative of a genome of an organism, to a solid support such as amicrochip, glass slide, or the like, in a defined pattern. By contactingthe microarray with a nucleic acid sample obtained from a cell ofinterest, and detecting those polynucleotides expressed in the cell thathybridize specifically to complementary sequences on the chip, thepattern formed by the hybridizing polynucleotides allows theidentification of clusters of genes that are expressed in the cell.Furthermore, when each polynucleotide linked to the solid support isknown, the identity of the hybridizing sequences from the nucleic acidsample can be identified.

Microarray technology provides a means to identify coordinate geneexpression simply by comparing patterns of hybridization. For example,by comparing the hybridization pattern of nucleic acid moleculesobtained from cells of an individual suffering from a disease with thenucleic acids obtained from the corresponding cells of a healthyindividual, clusters of genes that are differentially expressed can beidentified. The identification of such differentially expressed genesprovides a means to identify new genes, and provides insight as to thepattern of gene expression that occurs in a normal organism or in anorganism suffering from a pathologic condition.

Microarray technology further allows the identification of clusters ofgenes that are coordinately regulated and that encode proteins common toparticular intracellular pathways. Thus, microarray technology has beenused to determine that proteins involved in metabolic pathways such asphotosynthesis in plants and cuticle formation and lipid metabolism infruit flies are encoded by genes that are coordinately regulated and,further, that such coordinate expression is circadian regulated, i.e.,cycles with approximately twenty-four hour periodicity corresponding today and night.

Despite the large number of genes that have been identified as circadianregulated, the key gene or genes that determine the sleep/wake cycle inhumans have remained elusive. The identification of such a gene or geneswould provide a target for drugs that could be used, for example, tospecifically induce sleep in individuals suffering from insomnia,without causing undesirable side effects common to currently used drugs.Thus, a need exists to identify genes that encode proteins that regulatethe sleep/wake cycle. The present invention satisfies this need andprovides additional advantages.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that the cyclicexpression of a calcium dependent potassium channel (BK channel)correlates to locomotor activity and anticipates the sleep/wake cycle.Accordingly, the present invention provides screening assays foridentifying agents that modulate circadian regulated locomotor activityand agents that modulate the sleep/wake cycle in a subject; agentsidentified using such methods; and methods of utilizing such agents tomodulate locomotor activity or the sleep/wake cycle in a subject.

The present invention relates to a method of identifying an agent thatcan modulate the sleep/wake cycle in a subject. Such a method can beperformed, for example, by contacting a test system, which includes a BKchannel, with an agent suspected of having the ability to modulate thesleep/wake cycle in the subject; detecting a change in activity of theBK channel in the presence of the agent as compared to the activity ofthe BK channel in the absence of the agent, thereby identifying an agentthat modulates BK channel activity; administering the agent thatmodulates BK channel activity to a test subject; and detecting a changein the sleep/wake cycle of the test subject due to administration of theagent that modulates BK channel activity, thereby identifying an agentthat can modulate the sleep/wake cycle in a subject. The BK channel usedin such a screening assay can be in an isolated form; can be containedin a membrane, which can be a synthetic membrane or an isolatednaturally occurring membrane; or can be contained in a membrane of anintact cell, preferably ex vivo, including in a membrane of a cell thatnormally expresses the BK channel or in a membrane of a cell that hasbeen genetically modified to express the BK channel.

The BK channel can be a BK channel of any species, preferably aeukaryotic species, including an invertebrate such as an insect or anematode, or a vertebrate such as an amphibian, avian or mammalianspecies. In one embodiment, the BK channel is a Drosophila slowpoke(slo) polypeptide. In another embodiment, the BK channel is an orthologof a Drosophila slo polypeptide, for example, a mammalian ortholog suchas mouse slo (Kcnma1) polypeptide or a human slo polypeptide. In stillanother embodiment, the BK channel is a mutant BK channel, for example,a mutant Drosophila slo polypeptide.

A test system for practicing a method of the invention can contain asubstantially purified BK channel polypeptide, and contacting with theagent can be performed in vitro, for example, in a test tube, in a wellof a plate, or in a circumscribed position on a microchip. The BKchannel also can be contained in and traverse a membrane. In oneembodiment, the membrane is a synthetic membrane, for example, aliposome or synthetic lipid bilayer, having a first side and a secondside, which can, but need not, be an interior side and an exterior side,wherein the BK channel traverses the membrane. In another embodiment,the membrane is a cell membrane, which is isolated from a cell. In oneaspect of this embodiment, the cell membrane is obtained from a cellthat naturally expresses the BK channel, which can be a wild type or amutant BK channel, for example, a cell membrane isolated from a musclecell or a nerve cell of a eukaryotic organism such as a mammal. Inanother aspect of this embodiment, the cell membrane is isolated from acell that has been genetically modified to express a heterologous BKchannel. The heterologous BK channel can be the only BK channelexpressed in the cell membrane, or can be co-expressed with anendogenous BK channel, either or both of which can be a mutant BKchannel.

In another embodiment, a method of the invention is practiced using acell that is delimited by a cell membrane, wherein the BK channel isexpressed in the cell membrane. The BK channel can be an endogenous BKchannel, or can be a heterologous BK channel expressed, for example,from a polynucleotide introduced into the cell or into a cell from whichthe genetically modified cell is derived. A heterologous BK channel canbe transiently expressed in the genetically modified cell, or theencoding polynucleotide can be stably maintained in the cell. In oneembodiment, the cell expressing the BK channel is contacted with a testagent ex vivo, for example, in a cell culture or in a tissue or organculture. Thus, the cell can be a mammalian cell such as a human nervecell or muscle cell, which naturally expresses a BK channel, or can be aXenopus oocyte, which is genetically modified to express a BK channel,for example, a Drosophila slo polypeptide or a homolog, ortholog,paralog, or variant thereof. In another embodiment, the cell iscontacted in an organism in situ, wherein the organism can, but neednot, be a transgenic organism containing cells expressing, for example,a heterologous BK channel.

In a screening assay of the invention, the test system, which can be areaction mixture containing an isolated BK channel polypeptide or anisolated cell membrane, or an intact cell, can further contain a BKchannel binding protein. The BK channel binding protein can be, forexample, a Drosophila slo binding protein (slob) or a homolog, ortholog,paralog, or variant thereof. In some organisms such as mammals, the BKchannel can be in the form of a heterodimer, including an I subunit,which is an ortholog of Drosophila slo and comprises the pore formingunit, and a ∂ subunit, which has a regulatory activity. Accordingly, theBK channel binding protein in a test system can be 4 subunit of a BKchannel, for example, a human ∂ subunit.

BK channel activity, including a change in BK channel activity due tocontact with a test agent, can be examined using any of various wellknown methods for measuring channel activity, including methods fordetecting voltage gated activity and methods of detecting calcium iongated activity. For example, BK channel activity can be detected usingan electrophysiological method such as a patch-clamp assay or a voltageclamp recording. BK channel activity also can be detected using a methodthat directly or indirectly detects passage of molecules through thechannel, for example, passage of a fluorescent dye such as fura-2 orindo-1 or of an ion such as rubidium ion, or a change in expression of areporter gene due to a change in the calcium ion or potassium ionlevels. BK channel activity also can be measured by detecting aconformational change in the BK channel structure that is indicative ofchannel activity, for example, a fluorescence resonance energy transferassay, or using a physical method such as Fourier transform infraredresonance spectroscopy, Raman spectroscopy, fluorescence polarization,or atomic force microscopy. Where the steps for identifying an agentthat modulates BK channel activity are adapted to a high throughputformat, the means for detecting a change in BK channel activity isselected accordingly.

In a method of the invention, an agent suspected of having the abilityto modulate the sleep/wake cycle is examined initially for the abilityto modulate BK channel activity, then an identified agent that modulatesBK channel activity is administered to a test subject to identify anagent that can modulate the sleep/wake cycle. An agent that modulatesthe sleep/wake cycle can be identified by detecting a change in thesleep/wake pattern of the subject, either in comparison to thesleep/wake pattern in the subject prior to administration of the testagent, or in comparison to a sleep/wake pattern characteristic of anormal population comprising the subject. Routine statistical analysescan be used to determine a sleep/wake cycle characteristic of a subject(prior to and/or after treatment with a test agent) or of a normalpopulation.

An agent that modulates BK channel activity and is suspected of beingable to modulate the sleep/wake cycle can be administered to the testsubject in any convenient manner, including, for example, orally or byinjection. The test subject can be any subject suitable for testing theagent for the ability to modulate the sleep/wake cycle. In general, asuitable test subject expresses BK channels in muscle cells, nervecells, or both, wherein the BK channels are substantially similar tothose used for identifying an agent that modulates BK channel activity.The test subject can naturally express the BK channel, which can be awild type or mutant BK channel, or can be genetically modified toexpress the BK channels, for example, a transgenic non-human subjectcontaining cells expressing the heterologous BK channel.

An agent that is suspected of having the ability to modulate thesleep/wake cycle and that is to be examined according to a method of theinvention can have any chemical structure. As such, the agent can be apolynucleotide, a peptide, a peptidomimetic, a peptoid, or a smallorganic molecule. Since aspects of a method of the invention areadaptable to high throughput formats, the agents to be screened caninclude a library of agents, which can be a random library, variegatedlibrary, or the like. An agent that modulates the sleep/wake cycle canact by increasing BK channel activity or by decreasing BK channelactivity, including by increasing or decreasing the activity of a mutantBK channel. Accordingly, the present invention also provides an agentthat modulates the sleep/wake cycle in a subject, wherein the agent isidentified according to a method as disclosed herein.

The present invention also relates to a method of identifying an agentthat can modulate circadian regulated locomotor activity in a subject.Such a method can be performed, for example, by contacting a test systemcontaining a BK channel with an agent suspected of having the ability tomodulate circadian regulated locomotor activity in the subject;detecting a change in activity of the BK channel in the presence of theagent as compared to the activity of the BK channel in the absence ofthe agent, thereby identifying an agent that modulates BK channelactivity; administering the agent that modulates BK channel activity toa test subject; and thereafter detecting a change in circadian regulatedlocomotor activity of the test subject, thereby identifying an agentthat can modulate circadian regulated locomotor activity in a subject.

The present invention further relates to a method of modulating thesleep/wake cycle in a subject by administering an agent that modulatesBK channel activity to the subject. The subject can be any subject inwhich it is desired to modulate the sleep/wake cycle. For example, thesubject to be treated can be suffering from an acute or chronic sleepdisorder, wherein administration of the agent modulates the sleep/wakecycle of the subject so as to more closely approximate the sleep/wakecycle of a normal population to which the subject belongs. The subjectto be treated also can be one wishing to change his or her otherwisenormal sleep/wake cycle, for example, in preparation for travel so as toavoid jet lag, or to facilitate adjustment to non-standard work hourssuch as a night shift.

The agent can be administered to a subject by any convenient means,including, for example, orally in the form of a tablet or a capsule, oras a component of food or water to which the subject has access. Theagent also can be administered, for example, via a pump or can beformulated in a time-released form, thus providing a means to maintainthe agent at a desired level over a period of time. A time-released formof the agent can be contained, for example, in a matrix, which can beadministered to a subject intradermally, subcutaneously, orintramuscularly.

The present invention also provides a method of modulating circadianregulated locomotor activity in a subject by administering an agent thatmodulates BK channel activity to the subject. The subject can be anysubject for which it desired to regulate locomotor activity in acircadian manner, including, for example, a human subject suffering froman anxiety disorder, or a herd of farm animals so as to moreconveniently control the herd.

The present invention also relates to the identification of specificcellular proteins, in addition to the BK channel, that cycle in acircadian regulated manner in the suprachiasmatic nucleus and in theliver (see Table 5). The rhythmic cycling of these proteins in both thehead and body indicates that the proteins are representative of basiccomponents of a circadian clock. As such, these rhythmically cyclingproteins, which include, for example, receptors, including hormonereceptors, and enzymes, including kinases and phosphatases, provideadditional targets useful in screening assays to identify agents thatcan modulate the sleep/wake cycle. In addition, the regulatory elementsof the genes encoding these clock regulated proteins provide targetsuseful in screening assays for identifying agents that regulate theexpression of the clock regulated proteins. In particular, the clockregulated proteins of Table 5 (below), and the regulatory elements ofthe genes encoding these proteins, can be used in high throughputscreening assays, for example, wherein two or more of the proteins (orregulatory elements) are examined in parallel to identify agents thatregulate the activity (or expression) of one or a plurality of theproteins.

DETAILED DESCRIPTION OF THE INVENTION

Most organisms have endogenous biological clocks that coordinatephysiology and behavior to adapt to diurnal changes in the environment.In mammals, the suprachiasmatic nucleus (SCN) is the anatomical site ofa master pacemaker that regulates rhythmic processes throughout thebody. Recent work indicates that peripheral circadian clocks also exist,suggesting they may exert proximal regulation of physiology specific totheir target tissues. In Drosophila, a number of key processes such asemergence from the pupal case, locomotor activity, feeding, and aspectsof mating behavior are under circadian regulation. Although themechanisms by which the molecular oscillations take place are generallyunderstood, a clear link between gene regulation and downstreambiological processes has not previously been established.

An oligonucleotide-based high density array that interrogates geneexpression changes on a whole genome level was used to identify clockcontrolled output genes in Drosophila and in mice. As disclosed herein,a variety of physiological processes ranging from protein stability anddegradation, signal transduction, heme metabolism and detoxificationwere found to be under circadian transcriptional regulation inDrosophila (Example 1; see, also, Panda et al., Nature 417:329-335,2002a, which is incorporated herein by reference). A comparison ofrhythmically expressed genes in the fly head and body revealed that theclock has adapted its output functions to the needs of each particulartissue, thus indicating that tissue-specific gene expression issuperimposed on clock control of gene expression. A cycling calciumdependent potassium channel protein, slowpoke (slo) was identified asproviding a key step in linking the transcriptional feedback loop torhythmic locomotor behavior. As disclosed herein, expression of slocorrelated with regulation of the sleep/wake cycle in Drosophila.

Examination of circadian output genes in the mouse SCN, which includesthe central clock, and in mouse liver, which is an important regulatorof physiology, revealed that approximately 10% of detectably expressedtranscripts in both tissues were under circadian control, and thatalmost all of these output genes were specific to either the SCN orliver (Example 2). In addition, twenty proteins were identified thatcycled in both the head and body (Table 5). The cycling of theseproteins in the head and body indicates they represent basic clockcomponents. Genes encoding proteins in rate-limiting steps in pathwaysinvolved in endocrine regulation of physiology, energy metabolism, andthe redox state of the cell, and genes coding for both intracellular andextracellular signaling components were clock regulated. Remarkably,clock-regulated expression of the Kcnma1 gene, which encodes a calciumactivated potassium channel orthologous to that encoded by theDrosophila slo gene, also was identified in mouse SCN. These resultsindicate that cyclic potassium channel activity is involved in thecoordination of the rhythmic locomotor activity associated with thesleep/wake cycle, in eukaryotic organisms.

Clusters of genes involved in specific various biological pathways wereidentified as being coordinately expressed in a circadian-regulatedmanner in Drosophila and in the mouse (see Examples 1 and 2).Furthermore, when circadian-regulated expression of gene clusters wasexamined in the fly head as compared to fly bodies, only a fewtranscripts cycled in both tissues (Example 1; see, also, Ceriani etal., J. Neurosci. 22:9305-9319, 2002, which is incorporated herein byreference). Similar results were obtained when cycling transcripts inthe mouse SCN were compared to those cycling in mouse liver (Example 2;see, also, Table 5; see, also, Panda et al., Cell 109:307-320, 2002b,which is incorporated herein by reference). However, while commoncycling transcripts in central (head) as compared to peripheral tissueswere rare, many transcripts that cycled, for example, in fly heads alsowere expressed in the fly bodies, indicating that differentialtranscriptional regulation occurs with respect to these genes.

One of the fly genes identified to be circadian-regulated in fly heads,but not in fly bodies, was that encoding the slowpoke binding protein,slob, which binds to the calcium ion-dependent voltage gated potassiumchannel, slowpoke (see Example 1). McDonald and Rosbash (supra, 2001)reported circadian cycling of slob expression, and suggested thatcycling slob, through its interaction with slo, could give rise tocircadian oscillations in potassium channel activity. The authorsfurther suggested that such circadian oscillations in potassium channelactivity could affect resting membrane potential, which, in turn, wouldresult in calcium ion oscillations that may underlie oscillations inneuropeptide staining reported in lateral neuron termini (Id.).Circadian cycling of slob expression also was reported by Claridge-Changet al. (supra, 2001), who suggested that oscillating slob protein,through its interaction with slo, may be involved in rhythmic control ofsynaptic function, including synaptic plasticity, a process that mayrequire sleep. Claridge-Chang et al. further demonstrated by in situhybridization that slob mRNA expression occurred in the developingmushroom body of the fly larval brain, and corresponded with the regionof larval brain receiving projections of lateral neurons (LNs), whichcomprise the circadian pacemaker cells, and, based on theseobservations, suggested that innervating LNs may be required for cyclingslob expression (Id.).

Neither McDonald and Rosbash (supra, 2001) nor Claridge-Chang et al.(supra, 2001) reported whether expression of slo mRNA correlated withslo protein expression or whether slo protein levels cycle in fly brain.It is well recognized that mRNA expression does not necessarilycorrelate with translation of an encoded protein in cells, and even whenmRNA is translated, there is not necessarily a correlation between thelevel of mRNA in the cells and the amount of protein generated. Forexample, the mammalian protein HIF-1I is constitutively expressed at themRNA level, however, HIF-1I protein only is apparent following exposureto low oxygen conditions (Proc. Natl. Acad. Sci., USA 92:5510-5514,1995). Furthermore, neither of the references correlate slo mRNAexpression with rhythmic locomotor activity in anticipation of dusk anddawn or with regulation of the sleep/wake cycle.

As disclosed herein, slob mRNA cycled robustly in the heads of fliesexposed to either entrained (LD) or free running (DD) conditions,peaking at about ZT18, but did not cycle in clk^(jrk) flies, which aremutants that have impaired clock function (Example 1). Based on thisresult, expression of slo was examined and found to oscillate in phasewith slob, with a peak expression at ZT20 (see Ceriani et al., supra,2002; FIGS. 4A to 4C). Immunocytochemical analysis of whole fly brainmounts revealed that the slo protein was localized in a subset of theventral LNs. In wild type flies, cycling of slo correlated withlocomotor activity, which increased in anticipation of dawn and dusk. Incomparison, flies containing slo mutations failed to show a change inactivity in anticipation of dusk and dawn, even though total activityfor wild type and slo mutant flies was approximately the same (seeCeriani et al., supra, 2002; FIG. 5). As such, the present disclosureextends previous observations by demonstrating that slo, regulateschanges in locomotor activity in anticipation of dawn and dusk, thusindicating that slo is a key regulator of the sleep/wake cycle.Furthermore, clock regulated expression of the Kcnma1 gene, which is aslo ortholog in mice, paralleled that found in Drosophila (see Example2), indicating that the factors regulating the sleep/wake cycle areevolutionarily conserved. Accordingly, the present invention providesmethods of modulating the sleep/wake cycle and methods of modulatingcircadian regulated locomotor activity in an individual by increasing ordecreasing calcium activated potassium channel (“BK channel”) levels oractivity; drug screening assays, which allow the identification ofagents that increase or decrease BK channel activity and, therefore, canmodulate circadian regulated locomotor activity or the sleep wake cycle;and agents identified using such a screening assay.

In addition to clock regulated BK channel gene expression, which islocalized to the head, several genes that cycle in a circadian regulatedmanner in both the suprachiasmatic nucleus and liver of mice wereidentified (Table 5). The sequences of the polypeptides can be obtainedby searching for the appropriate identifier in the NCBI database(“Refseq” in Table 5) or in the Unigene database (“Unigene” in Table 5),which also provides ready access to orthologs of the listed mousepolypeptides. The more generalized rhythmic cycling of the clockregulated proteins listed in Table 5 indicates that they represent basiccomponents of the circadian clock. As such, these rhythmically cyclingproteins, as well as the genes encoding them (particularly theregulatory elements of such genes), provide additional targets useful inscreening assays to identify agents that can modulate the circadianclock in an organism.

Screening assays of the invention are exemplified herein with referenceto the BK channel. It will be recognized, however, that screening assaysof the invention also can be performed using one or more of the proteinsshown in Table 5, except that the methods will utilize an assay usefulfor detecting a change in the activity or function of the particularprotein or proteins used in the assay. For example, where the proteinused in a screening assay of the invention is a kinase such as caseinkinase 1, alpha 1 or adenylate kinase or a phosphatase such as proteintyrosine phosphatase, non-receptor type 16 or protein tyrosinephosphatase 4a1 (see Table 5), the screening assay will comprisecontacting the kinase or phosphatase with a substrate for the kinase orphosphatase, and an agent to examined for the ability to modulate thekinase or phosphatase activity, wherein a change in such activity due tothe agent identifies the agent as one that can modulate a circadianfunction in a subject expressing the protein in a clock regulatedmanner. In one embodiment, a screening assay of the invention comprisesa high throughput assay, wherein at least two of the polypeptides ofTable 5, including, if desired, the mouse BK channel and/or the mousePer2 gene product, or at least two regulatory elements comprising the 5′untranslated regions of the gene sequences encoding such polypeptides,can be contacted in parallel with one or more agents to be examined foran ability to modulate the activity of one or more of the polypeptides(or regulatory elements), thus providing a means to identify an agentthat modulates the activity or level of expression of one or more clockregulated proteins. If desired, such an agent then can be examined, forexample, for an ability to modulate the sleep/wake cycle or other clockregulated biochemical or physiological activity of a subject.

Accordingly, in one embodiment, the a screening assay of the inventionprovides means of identifying agents that can modulate the sleep/wakecycle in a subject. As exemplified herein, a screening assay of theinvention can be performed, for example, by contacting a test system,which includes a BK channel, with an agent suspected of having theability to modulate the sleep/wake cycle in the subject; detecting achange in activity of the BK channel in the presence of the agent ascompared to the activity of the BK channel in the absence of the agent,thereby identifying an agent that modulates BK channel activity;administering the agent that modulates BK channel activity to a testsubject; and detecting a change in the sleep/wake cycle of the testsubject due to administration of the agent that modulates BK channelactivity due to administration of the agent.

In another embodiment, a screening assay of the invention provides ameans of identifying agents that can modulate circadian regulatedlocomotor activity in a subject. Such a method can be performed, forexample, by contacting a test system containing a BK channel with anagent suspected of having the ability to modulate circadian regulatedlocomotor activity in the subject; detecting a change in activity of theBK channel in the presence of the agent as compared to the activity ofthe BK channel in the absence of the agent, thereby identifying an agentthat modulates BK channel activity; administering the agent thatmodulates BK channel activity to a test subject; and detecting a changein circadian regulated locomotor activity of the test subject due toadministration of the agent.

As used herein, the term “BK channel” or “calcium activated potassiumchannel” refers to the high conductance channel that is present inneuronal tissue and smooth muscle of eukaryotic organisms and is gatedby intracellular calcium ion concentration and membrane potential. Forpurposes of the present invention, a BK channel comprises at least aDrosophila slo polypeptide, or a homolog, ortholog, or paralog thereof(collectively “wild type” slo or BK channel), or a variant of a wildtype slo polypeptide (e.g., mutant). Such channels, which, in organismssuch as mammals, can contain an I subunit and a ∂ subunit, also arereferred to as “maxi-K channels”, enable efflux of potassium ions whenopened due to an increase in the intracellular calcium ion concentrationor membrane depolarization (change in potential). A BK channel useful ina drug screening assay of the invention can be a BK channel of anyspecies, preferably a eukaryotic species, including an invertebrate suchas an insect or a nematode, or a vertebrate such as an amphibian, avianor mammalian species.

BK channels are well known in the art and exemplified by those encodedby the Drosophila slowpoke (slo) gene, as well as by eukaryoticorthologs of slo, including mammalian slo (also referred to as Kcnma1)gene products, for example, the mouse slo (mslo) and human slo (hslo)orthologs. The nucleotide and amino acid sequences of Drosophila slo(Atkinson et al., Science 253:551, 1991; Adelman et al., Neuron 9:209,1992; GenBank Acc. No. NM_(—)079762, each of which is incorporatedherein by reference) are well known, as are those of orthologs such asthe mouse Kcnma1 ortholog (Butler et al., Science 261:221, 1993;Pallanck and Ganetzsky, Hum. Mol. Genet. 3:1239, 1994; GenBank Acc. No.NM_(—)010610, each of which is incorporated herein by reference), humanKcnma1 (see Butler et al., supra, 1993; Pallanck and Ganetzsky, supra,1994; see, also, Dworetzky et al., Brain Res. Mol. Brain. Res. 27:189,1994; Tsang-Crank et al., Neuron 13:1315, 1994; GenBank Acc. No.NM_(—)002247, each of which is incorporated herein by reference), ratKcnma1 (see, for example, GenBank Acc. No. NM_(—)031828, which isincorporated herein by reference); rabbit Kcnma1 (see, for example,GenBank Acc. No. AF321818, which is incorporated herein by reference).In view of the conserved sequence homology of the exemplified Drosophilaand mammalian BK channel nucleotide and amino acid sequences, it will berecognized that other wild type slo polynucleotides and variants thereofreadily can be identified and used in the methods of the invention.

The BK channel (or other clock regulated gene product) used in ascreening assay of the invention can be in an isolated form, forexample, a BK channel expressed from a recombinant nucleic acid orgenerated using an in vitro translation or coupledtranscription/translation reaction. An isolated BK channel also can beobtained from cells that normally express the channel using routinemethods for isolating a polypeptide from a membrane fraction of cells orcan be generated using chemical synthesis methods. As used herein, theterm “substantially purified” or “isolated”, when used in reference to apolypeptide or a polynucleotide, means that the polypeptide orpolynucleotide is in a form other than that in which it exists innature. In general, an isolated polypeptide or polynucleotide isrelatively free of materials with which it is naturally associated within a cell. For example, a substantially purified clock regulated geneproduct such as a BK channel can comprise at least about 10% of amixture, generally at least about 25% of a mixture, usually at leastabout 50% of a mixture, and particularly about 90% or more of a mixturecontaining the polypeptide. A determination that a polypeptide or apolynucleotide is substantially purified can be made using well knownmethods, for example, by performing electrophoresis and identifying theparticular molecule as a relatively discrete band. A substantiallypurified polynucleotide, for example, can be obtained by cloning thepolynucleotide, or by chemical or enzymatic synthesis. A substantiallypurified polypeptide can be obtained, for example, by a method ofchemical synthesis, or using methods of protein purification, followedby proteolysis and, if desired, further purification by chromatographicor electrophoretic methods.

It should be recognized, however, that an isolated BK channelpolypeptide, for example, can be added to a reaction mixture or that anisolated polynucleotide encoding a BK channel polypeptide can beintroduced into a cell. Nevertheless, the polypeptide or polynucleotideis considered to be (or have been) substantially purified because it isnot in the form in which it exists in nature. Methods for isolating apolypeptide are well known and include, for example, extraction,precipitation, ion exchange chromatography, affinity chromatography, andgel filtration methods, including combinations of such methods. Forexample, a BK channel can isolated by affinity chromatography using anantibody or other protein that specifically binds the BK channel. BKchannel binding proteins are disclosed herein and well known in the art.

A test system for practicing a method of the invention can contain asubstantially purified BK channel polypeptide, and contacting with theagent can be performed in vitro, for example, in a test tube, in a wellof a plate, or in a defined position on a microchip, thus allowing forhigh throughput screening of agents suspected of having the ability tomodulate BK channel activity. Such an in vitro reaction generally isperformed in an aqueous solution, which can contain buffers thatmaintain the reaction at a desired pH; salts such as those providingpotassium ions and calcium ions; and other reagents useful forperforming such a reaction.

A BK channel used in a method of the invention also can be contained ina membrane, including a synthetic membrane or an isolated naturallyoccurring membrane; or a membrane of an intact cell that normallyexpresses the BK channel or that has been genetically modified toexpress the BK channel. The membrane can be a synthetic membrane, forexample, a liposome or a synthetic lipid bilayer. Generally, though notnecessarily, a BK channel will traverse the cell membrane, in which casethe cell membrane has a first side and a second side. Depending on theassay being performed, the first and second side can be opposite side ofa surface formed by the membrane, or can be an interior side and anexterior side of a membrane that forms an enclosed volume.

A membrane containing a BK channel and useful in a method of theinvention also can be a cell membrane that has been isolated from acell. The cell membrane can be obtained from a cell that naturallyexpresses the BK channel, for example, a cell membrane isolated from amuscle cell or a nerve cell of a eukaryotic organism such as a mammal.The cell membrane also can be isolated from a cell that has beengenetically modified to express a heterologous BK channel. In a cellmembrane obtained from such a genetically modified cell, theheterologous BK channel can be the only BK channel expressed in the cellmembrane, or can be co-expressed with an endogenous BK channel.Furthermore, in this and other aspects of a screening method of theinvention, the BK channel can be a wild type BK channel or a mutant BKchannel (see, for example, Elkins et al., Proc. Natl. Acad. Sci., USA83:8415, 1986, which is incorporated herein by reference).

A drug screening assay of the invention also can be practiced using anintact cell, which is delimited by a cell membrane containing a BKchannel. The cell can be a muscle cell, nerve cell, kidney cell,epithelial cell, or other cell that expresses an endogenous BK channel,or can be cell that is genetically modified to express a heterologous BKchannel from a polynucleotide introduced into the cell or into a cellfrom which used in the assay is derived. As used herein, the term“genetically modified” refers to a cell containing a heterologouspolynucleotide that has been introduced into the cell using arecombinant DNA method. The term “heterologous” is used herein toindicate that a polynucleotide or polypeptide is not endogenous to acell (or isolated cell membrane) in which it is introduced or contained,or that the polynucleotide or polypeptide is part of a construct suchthat it is in a form other than it normally would be found in a cell. Assuch, a polynucleotide, for example, encoding a BK channel that isintroduced into a cell is heterologous with respect to the cell, as is apolypeptide expressed therefrom. It should be recognized that such aheterologous polynucleotide or polypeptide can be identical to anendogenous polynucleotide or polypeptide that also can naturally bepresent in the cell; for example, an expressible mouse slopolynucleotide can be introduced into mouse muscle cells that expressendogenous mouse slo polypeptide, such that the number of slopolypeptides expressed in the cell is increased, thus providing a meansto increase the sensitivity of a screening assay of the invention.

A heterologous polynucleotide, which can encode a wild type or mutant BKchannel polypeptide or BK channel binding protein, a reporterpolypeptide, or other polypeptide as desired, can be transientlyexpressed in the genetically modified cell, or can be stably maintainedin the cell. The polynucleotide can be contained in a vector, which canfacilitate manipulation of the polynucleotide, including introduction ofthe polynucleotide into a target cell. The vector can be a cloningvector, which is useful for maintaining the polynucleotide, or can be anexpression vector, which contains, in addition to the polynucleotide,regulatory elements useful for transcription and, where appropriate,translation of the polynucleotide. An expression vector can contain theexpression elements necessary to achieve, for example, sustainedtranscription of the encoding polynucleotide, or the regulatory elementscan be operatively linked to the polynucleotide prior to its beingcloned into the vector. For example, the polynucleotide can beoperatively linked to a tissue specific regulatory element, for example,a muscle cell specific regulatory element, such that expression of anencoded polypeptide is restricted to muscle cells. Muscle cell specificregulatory elements including, for example, the muscle creatine kinasepromoter (Sternberg et al., Mol. Cell. Biol. 8:2896, 1988, which isincorporated herein by reference) and the myosin light chainenhancer/promoter (Donoghue et al., Proc. Natl. Acad. Sci., USA 88:5847,1991, which is incorporated herein by reference) are well known in theart.

An expression vector (or the polynucleotide) generally contains orencodes a promoter sequence, which can provide constitutive, inducible,tissue specific, or developmental stage specific expression of theencoding polynucleotide, a poly-A recognition sequence, and a ribosomerecognition site or internal ribosome entry site, or other regulatoryelements such as an enhancer, which can be tissue specific. The vectoralso can contain elements required for replication in a prokaryotic oreukaryotic host system or both, as desired. Such vectors, which includeplasmid vectors and viral vectors such as bacteriophage, baculovirus,retrovirus, lentivirus, adenovirus, vaccinia virus, semliki forest virusand adeno-associated virus vectors, are well known and can be purchasedfrom a commercial source (Promega, Madison Wis.; Stratagene, La JollaCalif.; GIBCO/BRL, Gaithersburg Md.) or can be constructed by oneskilled in the art (see, for example, Meth. Enzymol., Vol. 185, Goeddel,ed. (Academic Press, Inc., 1990); Jolly, Canc. Gene Ther. 1:51, 1994;Flotte, J. Bioenerg Biomemb. 25:37, 1993; Kirshenbaum et al., J. Clin.Invest. 92:381, 1993; each of which is incorporated herein byreference).

Viral expression vectors can be particularly useful for introducing apolynucleotide into a cell, including, where desired, a cell in asubject. Viral vectors provide the advantage that they can infect hostcells with relatively high efficiency and can infect specific celltypes. For example, a polynucleotide encoding a BK channel polypeptidesuch as Drosophila slo, mouse slo, human slo, or the like, can be clonedinto a baculovirus vector, which then can be used to infect an insecthost cell, thereby providing a means to produce large amounts of theencoded slo polypeptide. The viral vector also can be derived from avirus that infects cells of an organism of interest, for example,vertebrate host cells such as mammalian, avian or piscine host cells.Viral vectors have been developed for use in particular host systems,particularly mammalian systems and include, for example, retroviralvectors, other lentivirus vectors such as those based on the humanimmunodeficiency virus (HIV), adenovirus vectors, adeno-associated virusvectors, herpesvirus vectors, vaccinia virus vectors, and the like (seeMiller and Rosman, BioTechniques 7:980, 1992; Anderson et al., Nature392:25, Suppl., 1998; Verma and Somia, Nature 389:239, 1997; Wilson, NewEngl. J. Med. 334:1185 (1996), each of which is incorporated herein byreference).

A polynucleotide encoding a clock regulated gene product such as a BKchannel, or encoding a polypeptide that specifically interacts with theclock regulated gene product and is required for or facilitates itsactivity, for example, a BK channel binding protein, or encoding areporter molecule, selectable marker, or the like, which can, but neednot, be contained in a vector, can be introduced into a cell by any of avariety of methods known in the art (Sambrook et al., Molecular Cloning:A laboratory manual (Cold Spring Harbor Laboratory Press 1989); Ausubelet al., Current Protocols in Molecular Biology, John Wiley and Sons,Baltimore, Md. (1987, and supplements through 1995), each of which isincorporated herein by reference). Such methods include, for example,transfection, lipofection, microinjection, electroporation and, withviral vectors, infection; and can include the use of liposomes,microemulsions or the like, which can facilitate introduction of thepolynucleotide into the cell and can protect the polynucleotide fromdegradation prior to its introduction into the cell. Accordingly, apolynucleotide can be introduced into a cell as a naked nucleic acidmolecule, can be incorporated in a matrix such as a liposome or aparticle such as a viral particle, or can be incorporated into a vector.The selection of a particular method will depend, for example, on thecell into which the polynucleotide is to be introduced, as well aswhether the cell is isolated in culture, or is in a tissue or organ inculture or in situ.

Introduction of a polynucleotide into a cell by infection with a viralvector is particularly advantageous in that it can efficiently introducethe nucleic acid molecule into a cell ex vivo or in vivo (see, forexample, U.S. Pat. No. 5,399,346, which is incorporated herein byreference). Moreover, viruses are very specialized and can be selectedas vectors based on an ability to infect and propagate in one or a fewspecific cell types. Thus, their natural specificity can be used totarget the polynucleotide contained in the vector to specific celltypes. As such, a vector based on an HIV can be used to infect T cells,a vector based on an adenovirus can be used, for example, to infectrespiratory epithelial cells, a vector based on a herpesvirus can beused to infect neuronal cells, and the like. Other vectors, such asadeno-associated viruses can have greater host cell range and,therefore, can be used to infect various cell types, although viral ornon-viral vectors also can be modified with specific receptors orligands to alter target specificity through receptor mediated events.

Generally, a polynucleotide encoding a clock regulated gene product, forexample, a BK channel, is introduced into a cell with a polynucleotideencoding a selectable marker, which provides a means to select cellsthat contain the introduced polynucleotide. Selectable markers include,for example, those that confer antimetabolite resistance, for example,dihydrofolate reductase, which confers resistance to methotrexate(Reiss, Plant Physiol. (Life Sci. Adv.) 13:143-149, 1994); neomycinphosphotransferase, which confers resistance to the aminoglycosidesneomycin, kanamycin and paromycin (Herrera-Estrella, EMBO J. 2:987-995,1983) and hygro, which confers resistance to hygromycin (Marsh, Gene32:481-485, 1984), trpB, which allows cells to utilize indole in placeof tryptophan; hisD, which allows cells to utilize histinol in place ofhistidine (Hartman, Proc. Natl. Acad. Sci., USA 85:8047, 1988);mannose-6-phosphate isomerase which allows cells to utilize mannose (WO94/20627); ornithine decarboxylase, which confers resistance to theornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine(DFMO; McConlogue, 1987, In: Current Communications in MolecularBiology, Cold Spring Harbor Laboratory ed.); and deaminase fromAspergillus terreus, which confers resistance to Blasticidin S (Tamura,Biosci. Biotechnol. Biochem. 59:2336-2338, 1995). In addition, reportermolecules can act as markers that facilitate identification of a plantcell containing the polynucleotide encoding the marker include, forexample, luciferase (Giacomin, Plant Sci. 116:59-72, 1996; Scikantha, J.Bacteriol. 178:121, 1996), green fluorescent protein (Gerdes, FEBS Lett.389:44-47, 1996), and numerous others as disclosed herein or otherwiseknown in the art.

A cell expressing a BK channel can be contacted with a test agent exvivo, for example, in a cell culture or in a tissue or organ culture. Asdisclosed herein, the cell can be a eukaryotic cell, for example, amammalian cell such as a human nerve cell or muscle cell, whichnaturally expresses a BK channel, or a cell that is genetically modifiedto express a BK channel, for example, a Xenopus oocyte, which can begenetically modified to express a wild type or variant slo polypeptide(see, for example, U.S. Pat. No. 5,637,470). The cell to be contactedalso can be present in situ in an organism, which can, but need not, bea transgenic organism containing cells expressing, for example, aheterologous BK channel.

In a screening assay of the invention, the test system, which can be areaction mixture containing an isolated BK channel polypeptide or anisolated cell membrane, or an intact cell, can further contain a BKchannel binding protein, for example, a Drosophila slo binding protein(slob), which is encoded by slob (GenBank Acc. No. AY060721; Schopperleet al., Neuron 20:565, 1998, each of which is incorporated herein byreference), or a homolog, ortholog or paralog of Drosophila slob, or avariant thereof. In some organisms such as mammals, the BK channel isformed as a heterodimer, including an I subunit, which is an ortholog ofDrosophila slo and comprises the pore forming unit, and a ∂ subunit,which has a regulatory activity and, for purposes of the presentinvention, is considered a BK channel binding protein. Thus, where ascreening assay of the invention utilizes a mammalian slo protein, forexample, a human slo protein, the assay can further include the BKchannel ∂ subunit, i.e., a human ∂ subunit. Nucleotide and amino acidsequences encoding mammalian BK channel ∂ subunits are well known (see,for example, U.S. Pat. No. 5,637,470; Meera et al., FEBS Lett. 382:84,1996, each of which is incorporated herein by reference).

Although mammalian BK channels can comprise a heterodimer, it should berecognized that inclusion of the BK channel binding protein (i.e., ∂subunit) is not necessary for practicing a screening assay of theinvention. For example, when mouse slo was expressed alone in Xenopusoocytes, large conductance, potassium ion selective channel activitycharacteristic of BK channels was observed, and the activity wassensitive to charybdotoxin (CbTX) and iberiotoxin (ITX), which areselective for BK channels (Butler et al., supra, 1993). In otherexperiments, oocytes genetically modified to express a human q subunit,alone, exhibited no measurable potassium currents different from thosein control oocytes using whole oocyte and patch-clamp recording methods,whereas oocytes expressing only the human I subunit (hslo) exhibitedlarge outward currents that were activated at positive membranepotentials, and blocked by CbTX and ITX (U.S. Pat. No. 5,637,470).Oocytes genetically modified to express the human I and human ∂ subunitsalso exhibited outward potassium currents that were blocked by CbTX andITX. The magnitudes of currents were similar to those observed inoocytes expressing only the I subunit. However, the outward currents inoocytes expressing the I and ∂ subunits were activated at more negativepotentials than oocytes expressing only the I subunit, and wereactivated by a BK channel activator that did not activate the channel inoocytes expressing only the I subunit (U.S. Pat. No. 5,637,470; see,also, Meera et al., supra, 1996). Thus, in a screening assay foridentifying an agent that modulates the activity of a BK channel thatcomprises an I subunit and a ∂ subunit in nature, there can beadvantages to practicing a method of the invention with a test systemcontaining the BK channel (I subunit) and BK channel binding protein (∂subunit). It will be recognized, however, that not all BK channelbinding proteins bind all BK channels. For example, Drosophila slobbinds Drosophila slo but does not bind mouse slo (Schopperle et al.,supra, 1998), whereas human slo binds the human 4 subunit and the bovine∂ subunit, both of which up-regulate the slo (I subunit) channelactivity (Meera et al., supra, 1996).

In view of the exemplified polynucleotides and encoded BK channel and BKchannel binding protein polypeptides, it will be recognized that wellknown procedures and algorithms based on identity (or homology) to theexemplified sequences can be used to identify homologs, orthologs, andvariants thereof useful in the screening methods of the invention (see,for example, U.S. Pat. No. 5,966,712, which is incorporated herein byreference). Such polynucleotides, for example, can be identified byperforming a BLASTN search using the Drosophila slo or murine kcnmalpolynucleotides as query sequences and selecting those substantiallysimilar sequences, for example, sequences having an E value ≦1×10⁻⁸.Such homologous, orthologous or variant polynucleotides can be usefulfor performing the screening assays of the invention.

As used herein, the term “substantially similar”, when used herein withrespect to a nucleotide sequence, means a nucleotide sequencecorresponding to a reference polynucleotide, wherein the correspondingsequence encodes a polypeptide having substantially the same structureand function as the polypeptide encoded by the reference polynucleotide.For purposes of the present invention, a reference (or query) sequenceis a polynucleotide encoding a BK channel or a BK channel bindingprotein. Desirably the substantially similar nucleotide sequence encodesthe polypeptide encoded by the reference nucleotide sequence. Thepercentage of identity between the substantially similar nucleotidesequence and the reference polynucleotide is at least 60%, generally atleast 75%, particularly at least 90%, preferably at least 95%, and morepreferably at least 99%. A nucleotide sequence “substantially similar”to a reference polynucleotide can selectively hybridize to the referencepolynucleotide, but not to an unrelated polynucleotide, underhybridization conditions such as provided by incubation in 7% sodiumdodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in2×SSC, 0.1% SDS at 50° C.; generally by incubation in 7% SDS, 0.5 MNaPO₄, 1 mM EDTA at 50° C. with washing in 1×SSC, 0.1% SDS at 50° C.;particularly by incubation in 7% SDS, 0.5 M NaPO₄, 1 mM EDTA at 50° C.with washing in 0.5×SSC, 0.1% SDS at 50° C.; preferably by incubation in7% SDS, 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in 0.1×SSC, 0.1%SDS at 50° C.; and more preferably by incubation in 7% SDS, 0.5 M NaPO₄,1 mM EDTA at 50° C. with washing in 0.1×SSC, 0.1% SDS at 65° C.

The term “substantially similar,” when used in reference to apolypeptide sequence, means that an amino acid sequence relative to areference (query) sequence shares at least about 65% amino acid sequenceidentity, generally at least about 75% amino acid sequence identity,particularly at least about 85%, preferably at least about 90%, and morepreferably at least about 95% or greater amino acid sequence identity.Generally, sequences having an E ≦10⁻⁸ are considered to besubstantially similar to a query sequence. Such sequence identity cantake into account conservative amino acid changes that do notsubstantially affect the function of a polypeptide. As such, homologs ororthologs of the Drosophila and murine circadian-regulated genes,particularly Drosophila slo and murine kcnmal, variants thereof, andpolypeptides (and encoding polynucleotides) substantially similar tothose exemplified herein can be used for practicing the methods of theinvention.

Homology or identity can be measured using sequence analysis softwaresuch as the Sequence Analysis Software Package of the Genetics ComputerGroup (University of Wisconsin Biotechnology Center, 1710 UniversityAvenue, Madison, Wis. 53705). Such software matches similar sequences byassigning degrees of homology to various deletions, substitutions andother modifications. The terms “homology” and “identity,” when usedherein in the context of two or more polynucleotide or polypeptidesequences, refer to two or more sequences or subsequences that are thesame or have a specified percentage of nucleotides or amino acidresidues, respectively, that are the same when compared and aligned formaximum correspondence over a comparison window or designated region asmeasured using any number of sequence comparison algorithms or by manualalignment and visual inspection.

For sequence comparison, one sequence generally acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

The term “comparison window” is used broadly herein to include referenceto a segment of any one of the number of contiguous positions, forexample, about 20 to 600 positions, for example, amino acid ornucleotide position, usually about 50 to about 200 positions,particularly about 100 to about 150 positions, in which a sequence maybe compared to a reference sequence of the same number of contiguouspositions after the two sequences are optimally aligned. Methods ofalignment of sequence for comparison are well known in the art. Optimalalignment of sequences for comparison can be conducted, for example, bythe local homology algorithm of Smith and Waterman (Adv. Appl. Math.2:482, 1981), by the homology alignment algorithm of Needleman andWunsch (J. Mol. Biol. 48:443, 1970), by the search for similarity methodof Person and Lipman (Proc. Natl. Acad. Sci., USA 85:2444, 1988), eachof which is incorporated herein by reference; by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.); or by manual alignment and visualinspection. Other algorithms for determining homology or identityinclude, for example, in addition to a BLAST program (Basic LocalAlignment Search Tool at the National Center for BiologicalInformation), ALIGN, AMAS (Analysis of Multiply Aligned Sequences), AMPS(Protein Multiple Sequence Alignment), ASSET (Aligned SegmentStatistical Evaluation Tool), BANDS, BESTSCOR, BIOSCAN (BiologicalSequence Comparative Analysis Node), BLIMPS (BLocks IMProved Searcher),FASTA, Intervals & Points, BMB, CLUSTAL V, CLUSTAL W, CONSENSUS,LCONSENSUS, WCONSENSUS, Smith-Waterman algorithm, DARWIN, Las Vegasalgorithm, FNAT (Forced Nucleotide Alignment Tool), Framealign,Framesearch, DYNAMIC, FILTER, FSAP (Fristensky Sequence AnalysisPackage), GAP (Global Alignment Program), GENAL, GIBBS, GenQuest, ISSC(Sensitive Sequence Comparison), LALIGN (Local Sequence Alignment), LCP(Local Content Program), MACAW (Multiple Alignment Construction &Analysis Workbench), MAP (Multiple Alignment Program), MBLKP, MBLKN,PIMA (Pattern-Induced Multi-sequence Alignment), SAGA (SequenceAlignment by Genetic Algorithm) and WHAT-IF (see, also, U.S. Pat. No.5,966,712). Such alignment programs can also be used to screen genomedatabases to identify polynucleotide sequences having substantiallyidentical sequences to a polynucleotide encoding a BK channelpolypeptide or BK channel binding protein.

A number of genome databases are available for comparison. Severaldatabases containing genomic information annotated with some functionalinformation are maintained by different organizations, and areaccessible on the world wide web via the internet, for example, at theURLs “wwwtigr.org/tdb”; “genetics.wisc.edu”;“genome-www.stanford.edu/˜ball”; “hiv-web.lan1.gov”; “ncbi.nlm.nih.gov”;“ebi.ac.uk”; “Pasteur.fr/other/biology”; and “genome.wi.mit.edu”. Inparticular, sequences and expression characteristics of the circadianregulated expression of Drosophila and mouse genes as disclosed hereinare accessible on the world wide web at the URL“expression.gnf.org/circadian”.

The BLAST and BLAST 2.0 algorithms using default parameters areparticularly useful for identifying polynucleotides encodingpolypeptides substantially similar to the exemplified BK channelpolypeptides and BK channel binding proteins (Altschul et al., NucleicAcids Res. 25:3389-3402, 1977; J. Mol. Biol. 215:403-410, 1990, each ofwhich is incorporated herein by reference). Software for performingBLAST analyses is publicly available on the world wide web through theNational Center for Biotechnology Information at the URL“ncbi.nlm.nih.gov”. This algorithm involves first identifying highscoring sequence pairs (HSPs) by identifying short words of length W inthe query sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul et al., supra, 1977, 1990). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectations (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff, Proc. Natl. Acad. Sci., USA 89:10915, 1989)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, for example, Karlin and Altschul,Proc. Natl. Acad. Sci., USA 90:5873, 1993, which is incorporated hereinby reference). One measure of similarity provided by BLAST algorithm isthe smallest sum probability (P(N)), which provides an indication of theprobability by which a match between two nucleotide or amino acidsequences would occur by chance. For example, a nucleic acid isconsidered similar to a references sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.2, more preferably less than about0.01, and most preferably less than about 0.001.

Protein and nucleic acid sequence homologies can be evaluated using theBasic Local Alignment Search Tool (“BLAST”). In particular, fivespecific BLAST programs can be used to perform the following task:

(1) BLASTP and BLAST3 compare an amino acid query sequence against aprotein sequence database;

(2) BLASTN compares a nucleotide query sequence against a nucleotidesequence database;

(3) BLASTX compares the six-frame conceptual translation products of aquery nucleotide sequence (both strands) against a protein sequencedatabase;

(4) TBLASTN compares a query protein sequence against a nucleotidesequence database translated in all six reading frames (both strands);and

(5) TBLASTX compares the six-frame translations of a nucleotide querysequence against the six-frame translations of a nucleotide sequencedatabase.

The BLAST programs identify homologous sequences by identifying similarsegments, which are referred to herein as “high-scoring segment pairs,”between a query amino or nucleic acid sequence and a test sequence whichis preferably obtained from a protein or nucleic acid sequence database.High-scoring segment pairs are preferably identified (aligned) by meansof a scoring matrix, many of which are known in the art. Preferably, thescoring matrix used is the BLOSUM62 matrix (Gonnet et al., Science256:1443-1445, 1992; Henikoff and Henikoff, Proteins 17:49-61, 1993,each of which is incorporated herein by reference). Less preferably, thePAM or PAM250 matrices may also be used (Schwartz and Dayhoff, eds.,“Matrices for Detecting Distance Relationships: Atlas of ProteinSequence and Structure” (Washington, National Biomedical ResearchFoundation 1978)). BLAST programs are accessible through the U.S.National Library of Medicine, for example, on the world wide web at theURL “ncbi.nlm.nih.gov”.

An agent that is suspected of having the ability to modulate BK channelactivity and, therefore, circadian regulated locomotor activity or thesleep/wake cycle (referred to generally herein as a “test agent”), canhave any chemical structure. As such, the agent can be a polynucleotide,a peptide, a peptidomimetic, a peptoid, a small organic molecule, andthe like. Furthermore, the screening methods of the invention areadaptable to high throughput formats and, therefore, conveniently allowthe examination of libraries of test agents, including combinatoriallibraries, which can be randomized, biased, or variegated (see, forexample, U.S. Pat. No. 5,837,500, which is incorporated herein byreference). Methods for preparing a combinatorial library of moleculesthat can be tested for a desired activity are well known in the art andinclude, for example, methods of making a phage display library ofpeptides, which can be constrained peptides (see, for example, U.S. Pat.No. 5,622,699; U.S. Pat. No. 5,206,347; Scott and Smith, Science249:386, 1992; Markland et al., Gene 109:13-19, 1991; each of which isincorporated herein by reference); a peptide library (U.S. Pat. No.5,264,563, which is incorporated herein by reference); a peptidomimeticlibrary (Blondelle et al., Trends Anal. Chem. 14:83, 1995; a nucleicacid library (O'Connell et al., Proc. Natl. Acad. Sci., USA 93:5883,1996; Tuerk and Gold, Science 249:505, 1990; Gold et al., Ann. Rev.Biochem. 64:763, 1995, each of which is incorporated herein byreference); an oligosaccharide library (York et al., Carb. Res., 285:99,1996; Liang et al., Science, 274:1520, 1996; Ding et al., Adv. Expt.Med. Biol., 376:261-269, 1995; each of which is incorporated herein byreference); a lipoprotein library (de Kruif et al., FEBS Lett., 399:232,1996, which is incorporated herein by reference); a glycoprotein orglycolipid library (Karaoglu et al., J. Cell Biol., 130:567, 1995, whichis incorporated herein by reference); or a chemical library containing,for example, drugs or other pharmaceutical agents (Gordon et al., J.Med. Chem., 37:1385, 1994; Ecker and Crooke, BioTechnology, 13:351,1995; each of which is incorporated herein by reference).

An agent suspected of having the ability to modulate BK channel activityand, therefore, circadian regulated locomotor activity and/or asleep/wake cycle in a subject can be a peptide. As used herein, the term“peptide” refers to a polymer comprising two or more amino acid residuesor amino acid analogs that are covalently linked by a peptide bond,which can be a modified peptide bond. For example, a peptide test agentcan contain one or more D-amino acids, or one or more amino acidanalogs, for example, an amino acid that has been derivatized orotherwise modified at its reactive side chain. Similarly, one or morepeptide bonds in the peptide can be modified, or the reactive group atthe amino terminus or the carboxy terminus or both can be modified. Suchpeptides can be modified, for example, to have improved stability to aprotease, an oxidizing agent or other reactive material the peptide canencounter in a biological environment, and, therefore, can beparticularly useful for administration to a subject, which can be a testsubject or a subject to be treated according to a method of theinvention. Conversely, if desired, peptides can be designed to havedecreased stability in a biological environment, for example, byincluding protease sensitive sites, such that the period of time thepeptide is active in the environment is reduced.

A test agent also can be a polynucleotide. As used herein, the term“polynucleotide” means a polymer of two or more deoxyribonucleotides orribonucleotides, or analogs thereof, that are linked together by aphosphodiester or other bond. As such, the terms include RNA and DNA,which can be a gene or a portion thereof, a cDNA, a syntheticpolydeoxyribonucleic acid sequence, or the like, and can be singlestranded or double stranded, as well as a DNA/RNA hybrid. Although theterm “polynucleotide” is used herein to include naturally occurringnucleic acid molecules such as those encoding BK channel polypeptides,polynucleotides useful as test agents generally are non-naturallyoccurring molecules, which can be prepared, for example, by methods ofchemical synthesis or by enzymatic methods such as by the polymerasechain reaction (PCR). Polynucleotides can be particularly useful asagents that modulate BK channel activity and, therefore, circadianregulated locomotor activity or sleep/wake cycle because nucleic acidmolecules having binding specificity for cellular targets, includingcellular polypeptides, exist naturally, and because synthetic moleculeshaving such specificity can be readily prepared and identified (see, forexample, U.S. Pat. No. 5,750,342, which is incorporated herein byreference).

The nucleotides comprising a polynucleotide can be naturally occurringdeoxyribonucleotides, such as adenine, cytosine, guanine or thyminelinked to 2′-deoxyribose, or ribonucleotides such as adenine, cytosine,guanine or uracil linked to ribose; or nucleotide analogs, includingnon-naturally occurring synthetic nucleotides or modified naturallyoccurring nucleotides. Such nucleotide analogs are well known in the artand commercially available, as are polynucleotides containing suchnucleotide analogs (Lin et al., Nucl. Acids Res. 22:5220 (1994);Jellinek et al., Biochemistry 34:11363 (1995); Pagratis et al., NatureBiotechnol. 15:68 (1997), each of which is incorporated herein byreference). The covalent bond linking the nucleotides of apolynucleotide can be a phosphodiester bond, or any of numerous othercovalent bonds, including a thiodiester bond, a phosphorothioate bond, apeptide-like bond or any other bond known to those in the art as usefulfor linking nucleotides to produce synthetic polynucleotides (see, forexample, Tam et al., Nucl. Acids Res. 22:977 (1994); Ecker and Crooke,BioTechnology 13:351360 (1995), each of which is incorporated herein byreference). The incorporation of non-naturally occurring nucleotideanalogs or bonds linking the nucleotides or analogs can be particularlyuseful where the polynucleotide is to be exposed to an environment thatcan contain a nucleolytic activity, including, for example, a tissueculture medium or upon administration to a living subject, since themodified molecules can be less susceptible to degradation.

A polynucleotide containing naturally occurring nucleotides andphosphodiester bonds, can be chemically synthesized or can be producedusing recombinant DNA methods, using an appropriate polynucleotide as atemplate. In comparison, a polynucleotide containing nucleotide analogsor covalent bonds other than phosphodiester bonds generally will bechemically synthesized, although an enzyme such as T7 polymerase canincorporate certain types of nucleotide analogs into a polynucleotideand, therefore, can be used to produce such a polynucleotiderecombinantly from an appropriate template (Jellinek et al., supra,1995).

An agent that modulates circadian regulated locomotor activity and/orthe sleep/wake cycle can act by increasing BK channel activity or bydecreasing BK channel activity, including by increasing or decreasingthe activity of a mutant BK channel. Increased or decreased BK channelactivity due to contact with a test agent can be examined using any ofvarious methods known in the art for measuring potassium channelactivity (see, for example, Meth. Enzymol.: Ion Channels (eds. Abelsonet al., Academic Press 1998), which is incorporated herein byreference). For example, BK channel activity can be examined by makingelectrophysiological recordings such as by performing patch-clampelectrophysiologic analysis of cells following stable or transienttransfection of cDNA molecules encoding slo and, if desired, slob ororthologs thereof, or voltage-clamp recording of Xenopus oocytes uponmRNA injection, cRNA, or cDNA injection. Extracellular or intracellularrecordings of transfected cells can be obtained.

Extracellular voltage recording requires measurements of smallbiological potentials, often less than a millivolt in amplitude. TheAxoclamp-2B, GeneClamp 500B and MultiClamp 700A microelectrodeamplifiers are useful for such experiments (Axon Instruments, Inc.;Union City Calif.). Voltage clamp allows measurement of membrane currentby monitoring the membrane voltage and injecting current to attain andmaintain the desired voltage. As such, a voltage-clamp amplifier isselected based on its ability to measure voltage, and passes current inorder to regulate the cellular voltage. Patch-clamp analysis utilizes ablunt pipette to isolate a patch of membrane. Patch-clamp recording canmeasure the individual ion channel currents that contribute to wholecell currents, and is compatible with current-clamp and voltage-clamprecording modes (see Axon Instruments, Inc., web site on the world wideweb at URL “axon.com”, information under “neurosciences” and “cellularneurosciences product lines”). In whole cell patch-clamping, the patchof membrane beneath the pipette is ruptured or otherwise made permeablesuch that currents passing through an entire cell membrane are recorded.This method is equivalent to intracellular recording with sharpmicroelectrodes, but has the advantage that it can be applied to cellsthat are very tiny or flat and would otherwise be very difficult toimpale. The magnitude of the transmembrane current varies greatlybetween cell types. As such, the use of two electrodes, one for passingcurrent and one for measuring voltage, is best for clamping large cellswith large currents (Id.). Voltage clamp amplifiers such as theAxoclamp-2B amplifier and GeneClamp 500B amplifier are particularlyuseful for measurements using the Two-Electrode Voltage-Clamp (TEVC)mode (Axon Instruments, Inc.).

Current-clamp amplifiers are designed to control the current and measurethe corresponding membrane voltage. It is common to pass current tostimulate a cell or modify its resting potential during intracellularvoltage recording. The Axoclamp-2B amplifier, GeneClamp 500B amplifier,and MultiClamp 700A amplifier (Axon Instruments, Inc.) can pass currentwhile in voltage-sensing (i.e., current-clamp) mode. The Axoclamp-2Bamplifier also allows for “discontinuous” recording modes, applicable toboth voltage clamp and current clamp. In this mode the instrumentdivides its time in passing current and recording voltage. The advantageof this mode is that the recording is free from the usual error due tothe voltage drop across the electrode resistance, and can be used with aconventional intracellular microelectrode. Ion-selective electrodes,voltammetry and constant-voltage amperometry also can be used to measurelevels and small changes in ion, neurotransmitter and hormoneconcentrations in tissues or in and near cells. These techniques requirethe ability to record small potentials and pass large currents.Ion-selective electrodes require differential input, low leakage currentand high-impedance voltage following. The electrochemical techniques ofvoltammetry and constant-voltage amperometry are used to measure fastchanges in neurotransmitter concentrations, and require a voltage-clampamplifier with a command voltage range extended to ±1V (AxonInstruments, Inc.).

Detection of BK channel activity in native systems or in recombinantexpression systems also can be examined using fluorescent dyes sensitiveto membrane potential or intracellular ions (including pH). The VoltageIon Probe Reader II system (VIPR II™ system) applies fluorescenceresonance energy transfer (FRET) technology to ion channel analysis(Aurora Biosciences Corp., San Diego Calif.; see web site on the worldwide web at the URL “aurorabio.com”, information under “Auroraplatforms” and “ion channel technology”). FRET is a distance-dependentinteraction between the electronic excited states of two dye molecules,and is useful for investigating biological events that produce changesin molecular proximity, including, for example, FRET between amembrane-bound donor molecule and a mobile, voltage-sensitive, acceptormolecule to detect membrane potential (“Voltage Sensor ProbeTechnology”; Aurora Biosciences Corp.). The VIPR II™ system can beperformed in a 96 well format or 384 well format and, therefore, isparticularly suitable for high throughput screening assays, allowing forthroughput of up to 40,000 samples per day under temperature controlledconditions. The system allows for dual emission fluorescence kineticreading in real time, and includes data collection and analysissoftware. The system can screen potassium and calcium gated ionchannels, reads approximately 5 mV changes in membrane potential inmilliseconds, and allows for single cell detection.

Aurora Biosciences Corp. also provides “Voltage Sensor Probes”technology, which combine rapid response and high sensitivity forreliable detection of changes in membrane voltage induced by modulationof ion channels (see web site on the world wide web at the URL“aurorabio.com”, information under “bioassay technologies” and “voltagesensitive probes”; see, also, “fluorescent probes”). Voltage SensorProbes technology uses two fluorescent molecules, including oxonol,which is a highly fluorescent, negatively charged, hydrophobic ion that“senses” the transmembrane electrical potential, and coumarin lipid,which binds specifically to one face of the plasma membrane andfunctions as a FRET donor to the voltage-sensing oxonol acceptormolecule. In response to changes in membrane potential, oxonol canrapidly redistribute between two binding sites on opposite sides of theplasma membrane. When oxonol moves to the intracellular plasma membranebinding site upon depolarization, FRET is decreased and results in anincrease in the donor fluorescence and a decrease in the oxonol emission(Id.).

Another fluorimetric system developed to measure channel activity is thefluorimetric imaging plate reader (FLIPR™) system (Molecular DevicesCorp.; Sunnyvale Calif.; see web site on world wide web at URL“moleculardevices.com”, search “FLIPR calcium assay”). The FLIPR™ systemconveniently can be performed in a 384 well high throughput format(FLIPR³⁸⁴) using a minimal sample volume. The FLIPR³⁸⁴ system canmonitor intracellular calcium, membrane potential, intracellular pH, andintracellular sodium from cells of a population in real time, providingmaximum versatility and the ability to identify a potential hit secondsafter it is added to the cell plate. Real-time, kinetic data alsoprovides additional pharmacological information for ranking relativepotencies of drugs, and gives information on the kinetics of thedrug-receptor interaction (Id.).

BK channel activity also can be examined using an ion flux assay, forexample, a rubidium ion efflux assay, which provides a functionalanalysis. Rubidium ion is similar in size and charge to potassium ionand confers similar permeability rates within the cell. BK channelactivity can be determined by quantifying rubidium ion levels in celllysate and supernatant fractions, wherein rubidium ion concentration inthe fractions is directly related to channel efflux. Rubidium ionconcentration can be determined using flame atomic absorptionspectroscopy method, which can be automated using, for example, an ICR8000 system (Aurora Biomed, Inc.; Vancouver BC; see, also, Aliphitiraset al., Soc. Biomol. Screening, 7th Ann. Conf., Poster Session 5, #5004,which is incorporated herein by reference).

Calcium ion flux also can be measured using dyes such as fura-2, indo-1,or derivatives thereof, which are UV light-excitable, ratiometriccalcium ion indicators (Molecular Probes, Inc.; Eugene Oreg.). Fura-2,for example, is useful for ratiometric imaging, and exhibits anabsorption shift that can be observed by scanning the excitationspectrum between 300 nm and 400 nm, while monitoring the emission atapproximately 510 nm. In comparison, indo-1 is useful flow cytometryanalysis. The emission maximum of indo-1 shifts from approximately 475nm in calcium ion-free medium to about 400 nm when the dye is saturatedwith calcium ion. The sodium and potassium salts of fura-2 and thepotassium salt of indo-1 are cell-impermeant probes that can bedelivered into cells by microinjection or using an influx pinocyticcell-loading reagent (Molecular Probes, Inc.; see web site on world wideweb at URL “molecularprobes.com”, information under “Handbook” and“chapter 20”). Quin-2 is another calcium ion indicator that has lowerabsorptivity and quantum yield values than the fura-2 and indo-1 and,therefore, requires higher loading concentrations, which can bufferintracellular calcium ion transients (Id.).

BK channel activity also can be examined using ion chelators or otherextracellular or intracellular reagents that exhibit a change inphysico-chemical properties upon potassium binding, or by detectingchanges in intracellular or extracellular pH levels (see, for example,U.S. Pat. No. 6,150,176; U.S. Pat. No. 6,140,132, each of which isincorporated herein by reference). In addition, changes in BK channelactivity can be detected by examining changes in the expression of genesthat are regulated by such changes, or by measuring the activity ofcalcium channels that are co-expressed with the BK channels andsensitive to membrane potential.

A change in BK channel activity also can be detected by examining theexpression of a reporter gene that is operatively linked to a generegulatory element that is responsive to the change in BK channelactivity. For example, glucagon contains a calcium response element,which regulates glucagon expression in response to calcium ionconcentration (Fürstenau et al., J. Biol. Chem. 274:5851, 1999, which isincorporated herein by reference). As such, the calcium response elementcan be operatively linked to a reporter gene, which, upon introductioninto a cell being examined according to a method of the invention,provides a means to detect changes in intracellular calcium ion due toan effect of a test agent on BK channel activity. Other gene regulatoryelements that are responsive to changes in calcium ion include, forexample, the cAMP response element and the serum response element (see,for example, Ginty, Neuron 18:183, 1997, which is incorporated herein byreference).

Reporter genes that can be operatively linked to a desired regulatoryelement are well known in the art and include, for example, a∂-lactamase, chloramphenicol acetyltransferase, adenosine deaminase,aminoglycoside phosphotransferase, dihydrofolate reductase, hygromycin-Bphosphotransferase, thymidine kinase, ∂-galactosidase, luciferase andxanthine guanine phosphoribosyltransferase polypeptide. Similarly,methods of detecting expression of such reporter genes are well knownand include, for example, methods of detecting a colorimetric,luminescent, chemiluminescent, fluorescent, or enzymatic activity due toexpression of the reporter polypeptide.

As used herein, the term “operatively linked” means that two or moremolecules are positioned with respect to each other such that they actas a single unit and effect a function attributable to one or bothmolecules or a combination thereof. For example, a polynucleotidesequence encoding a reporter polypeptide or the like can be operativelylinked to a regulatory element such as a calcium response element, inwhich case the regulatory element confers calcium inducible expressionon the reporter similarly to the way in which the regulatory elementwould effect, for example, expression of glucagon in a pancreatic isletcell. A first polynucleotide coding sequence also can be operativelylinked to a second (or more) coding sequence such that a chimericpolypeptide can be expressed from the operatively linked codingsequences. The chimeric polypeptide can be a fusion polypeptide, inwhich the two (or more) encoded peptides are translated into a singlepolypeptide, i.e., are covalently bound through a peptide bond; or canbe translated as two discrete peptides that, upon translation, canoperatively associate with each other to form a stable complex. Forexample, the fusion protein can comprise a fluorescent protein, whichcan be useful for constructing chimeric proteins for FRET analysis.

A change in BK channel activity also can be detected using a physicalmethod such as Fourier transform infrared analysis, atomic forcemicroscopy (Chen and Hansma, J. Struct. Biol. 131:44, 2000; Oesterheltet al., Science, 143, 2000; Stolz et al., J. Struct. Biol. 131:171,2000; Obregon et al., Biophys. J. 79:202, 2000, each of which isincorporated herein by reference), Raman spectroscopy, and the like, orby detecting a conformational change of the BK channel or a change inprotein-protein interaction such as between the BK channel and a BKchannel binding protein using a method such as FRET (U.S. Pat. No.6,342,379; U.S. Pat. No. 5,661,035, each of which is incorporated hereinby reference), B-RET, FIDA, FP, FCS. Such methods can be utilized fordetecting changes in BK channel in a cell or changes in a substantiallypurified BK channel in vitro.

Various drugs can act as potassium channel antagonists and, therefore,can be useful for confirming the accuracy and validity of a test systemcomprising a BK channel and as controls that can be run in parallel withtest agents. Such drugs include glyburide(1-{{{p-2-(5-chloro-o-anisamido)ethyl}phenyl}-sulfonyl}-3-cyclohexylurea),glipizide(1-cyclohexyl-3-{{{p-(2-(5-methylpyrazinecarboxamido)ethyl}phenyl}sulfonyl}urea)and tolbutamide (1-butyl-3-(p-methylbenzenesulfonyl)urea), which areused as anti-diabetic agents, and other antagonists that are used asClass III anti-arrhythmic agents and to treat acute myocardialinfarctions in humans. A number of naturally occurring toxins that blockpotassium channels, including apamin, IBX, CbTX, margatoxin,noxiustoxin, kaliotoxin, dendrotoxin(s), mast cell degranuating peptide,and ∂-bungarotoxin, also can be used for this purpose.

A screening assay of the invention provides a means to identify agentthat can modulate circadian regulated locomotor activity and/or thesleep/wake cycle of an individual. As used herein, the term “sleep/wakecycle” refers to a rhythmic pattern, in which sleep onset leads to aperiod of sleep, followed by awakening from sleep, and a period ofwakefulness, to be followed again by sleep onset, and so on. A normalsleep/wake cycle generally repeats in a circadian rhythm over a periodof about 24 hours, and is linked to the length of day and night, i.e.,the light/dark cycle. Reference herein to a “normal” or “typical”sleep/wake cycle means the sleep/wake cycle that is characteristic of apopulation of organisms in nature. For example, a normal sleep/wakecycle in humans includes sleep onset occurring about 4 to 5 hours aftersunset, followed by a period of sleep that ends with awakening about 1to 2 hours after sunrise (see, for example, Young and Kay, Nat. Rev.Genet. 2:702, 2001, which is incorporated herein by reference). Incomparison, a normal sleep/wake cycle for nocturnal organisms ischaracterized by sleep onset occurring at or about sunrise or shortlythereafter.

Although actual sleep/wake cycles can vary substantially amongindividuals of a population, observation over a period of time and ofspecific individual or of a representative population of individuals androutine statistical analyses can be used to determine a sleep/wake cyclecharacteristic of the specific individual (prior to and/or aftertreatment with a test agent) or of the population, which can be apopulation of otherwise healthy individuals or a population ofindividuals suffering from the same disorder, for example, insomnia. Itis recognized that in most populations, including humans, there will bea wide range of times “normal” individuals awaken or drowse to sleep.Nevertheless, any population of individuals will exhibit a normaldistribution of such times and, therefore, an mean and standarddeviation can be determined. For example, humans, on average, sleep forabout eight hours and are awake for about sixteen hours.

Circadian rhythms are nearly ubiquitous in nature, occurring inprokaryotes and eukaryotes. The processes under circadian control areequally diverse, ranging from human sleep/wake cycles to cell divisionin photosynthetic bacteria. The hallmark of these roughly 24 hourrhythms is their persistence under constant environmental conditions.This persistence is effected by the circadian clock, which is aninternal biochemical oscillator. The circadian clock allows an organismto anticipate daily changes in the environment such as the onset of dawnand dusk, thereby providing the organism with an adaptive advantage (Yanet al., Proc. Natl. Acad. Sci., USA 95:8660, 1998). As such, the term“circadian regulated locomotor activity” is used herein to refer torhythmic activity that is anticipatory of a daily change in theenvironment, particularly rhythmic activity that is anticipatory of theonset of dawn and dusk. In addition, the circadian clock regulates otherrhythmic activities, including, for example, hormonal rhythms, bloodpressure rhythms, body temperature rhythms, cholesterol production, andheme production. A normal pattern for a circadian regulated locomotoractivity or other circadian regulated rhythmic activity can bedetermined by observing individuals in a population and analyzing theresults using statistical methods, as discussed below with respect todetermining a normal sleep/wake cycle. Such methods provide a standardvalue, from which individuals exhibiting an arrhythmia in the locomotoractivity can be identified (see, also, Example 1).

In a screening assay of the invention, an agent suspected of having theability to modulate circadian regulated locomotor activity and/or thesleep/wake cycle is examined initially for the ability to modulate BKchannel activity, then agents that are identified as having the abilityto modulate BK channel activity are administered to test subjects toidentify those agents that also modulate circadian regulated locomotoractivity, the sleep/wake cycle, or both. The test subject can be anyorganism that expresses wild type or mutant BK channels in nerve cellsand muscle cells, including an organism that has been geneticallymodified to express such BK channels, for example, a transgenicnon-human organism. Generally, the test subject contains cells thatexpress substantially the same BK channels as were used in the initialtest system to identify agents that modify BK channel activity. However,the test subject also can contain cells that express a homolog, orthologor paralog of the BK channel used in the initial test system, or amutant or other variant of the BK channel. In addition, the screeningmethod can include one or more control subjects, which, for example,contain cells that express a BK channel that is not modulated by thetest agent, thus providing a means to confirm the specificity of anagent that is found to modulate the sleep/wake cycle or circadianregulated locomotor activity of a test subject.

A test subject is selected, in part, based on the characteristicsdesired of the test agent being examined. For example, if test agentsare being screened to identify those that can shift sleep and awakeningby a desired time (e.g., about four, six, eight or twelve hours), thetest subjects can be organisms that exhibit a normal sleep/wake cycle.In this respect, it should be recognized that many organisms, includingexperimental organisms such as mice, are primarily nocturnal. As such,while in humans, a “normal” sleep/wake cycle includes awakening aboutdawn (CT0) and drowsing to sleep after dusk (CT12), for example, atabout CT16 (assuming an average of 8 hours sleep), the cycle can bedifferent in an experimental organism that is used as a test subject foridentifying an agent that would be useful in humans. Nevertheless, theeffectiveness that an agent being tested in an experimental animal canhave in a human can be determined by accounting for these differences.Where a test agent is being examined, for example, to treat insomnia,the test subject can be selected based on having symptoms of insomnia,or can be placed in conditions that are not conducive to sleep, and theability of an agent that modulates BK channel activity to allow sleep tobegin at a normal time can be examined. Where a test agent is beingexamined for an ability to induce circadian regulated locomotoractivity, the test subject is selected based on a lack of such regulatedactivity, for example, a test subject exhibiting anxiety or hyperkineticactivity, or a subject exhibiting a circadian related sleep disordersuch as familial advance phase sleep disorder or familial delayed phasesleep disorder (see, for example, Sleep 22:616-623, 1999).

An agent that modulates the sleep/wake cycle can be identified bydetecting a change in the sleep/wake pattern of the subject, either incomparison to the sleep/wake pattern in the subject prior toadministration of the test agent, or in comparison to a sleep/wakepattern characteristic of a normal population comprising the subject.The agent can be administered to the test subject (or control subject)in any convenient manner, including, for example, orally or by injection(see, also, below). Using such a screening assay as disclosed herein,agents that modulate the sleep/wake cycle or circadian regulatedlocomotor activity in a subject by modulating BK channel activity can beidentified. Accordingly, the present invention provides agentsidentified by a screening assay. Such agents can be useful asmedicaments for treating a subject having, for example, a disorderaffecting the sleep/wake cycle.

The present invention also provides a method of modulating thesleep/wake cycle in a subject by administering an agent that modulatesthe activity of a clock regulated gene product, for example, BK channelactivity, to the subject. The subject can be any subject in which it isdesired to modulate the sleep/wake cycle. Generally, a subject to betreated is one suffering from an acute or chronic sleep disorder,wherein administration of the agent modulates the sleep/wake cycle ofthe subject so as to more closely approximate the sleep/wake cycle of anormal population to which the subject belongs. A sleep disorderamenable to treatment according to a method of the invention ischaracterized, in part, by an inability of the subject to establish aregular pattern of sleep, for example, insomnia or narcolepsy. A subjectto be treated also can be one wishing to change his or her otherwisenormal sleep/wake cycle. For example, a person preparing to travel to adifferent time zone, particularly a time zone that is at least aboutfour hours or six hours different from that in which the person isleaving, can take an agent that delays (or advances) the currentsleep/wake cycle a sufficient amount such that the person can avoid jetlag. Similarly, a person that works a night shift can benefit from achange in an otherwise normal sleep/wake cycle to one that accommodateshis or her work schedule. As such, a method of the invention can provideadvantages to the general public, including, for example, decreased riskof injuries due to tiredness during a night shift, or increasedproductivity of business person traveling to a distant time zone.

The present invention also provides a method of modulating circadianregulated locomotor activity in a subject by administering an agent thatmodulates BK channel activity to the subject. The subject can be, forexample, a person suffering from an anxiety or hyperkinetic disorder,wherein administration of agent that attenuates the locomotor activityassociated with the disorder provides a rhythmic decreased locomotoractivity. Such a rhythmic decreased locomotor activity can be timed, forexample, such that the subject can remain in a more “relaxed state”during work or school hours. A method of the invention also can beuseful for treating a herd of farm animals, such that individual animalsin the herd are not overly disruptive or such that the herd, in general,is more amenable to handling during desired times.

The agent can be administered to a subject by any convenient means,including, for example, orally in the form of a tablet or a capsule, oras a component of food or water to which the subject has access. Theagent also can be administered, for example, via a pump or can beformulated in a time-released form, thus providing a means to maintainthe agent at a desired level over a period of time. A time-released formof the agent can be contained, for example, in a matrix, which can beadministered to a subject intradermally, subcutaneously, orintramuscularly.

Generally, for administration to a subject, the agent is formulated in acomposition suitable for administration to the subject. As such, thepresent invention also provides compositions containing an agent, whichmodulates BK channel activity and further modulates the sleep/wake cycleor circadian regulated locomotor activity, in a pharmaceuticallyacceptable carrier. Pharmaceutically acceptable carriers are well knownin the art and include, for example, aqueous solutions such as water orphysiologically buffered saline or other solvents or vehicles such asglycols, glycerol, oils such as olive oil or injectable organic esters.A pharmaceutically acceptable carrier can contain physiologicallyacceptable compounds that act, for example, to stabilize or to increasethe absorption of the conjugate. Such physiologically acceptablecompounds include, for example, carbohydrates, such as glucose, sucroseor dextrans, antioxidants, such as ascorbic acid or glutathione,chelating agents, low molecular weight proteins or other stabilizers orexcipients. One skilled in the art would know that the choice of apharmaceutically acceptable carrier, including a physiologicallyacceptable compound, depends, for example, on the physico-chemicalcharacteristics of the agent and on the route of administration of thecomposition, which can be, for example, orally or parenterally such asintravenously, and by injection, intubation, or other such method knownin the art.

The agent can be incorporated within an encapsulating material such asinto an oil-in-water emulsion, a microemulsion, micelle, mixed micelle,liposome, microsphere or other polymer matrix (see, for example,Gregoriadis, Liposome Technology, Vol. 1 (CRC Press, Boca Raton, Fla.1984); Fraley, et al., Trends Biochem. Sci., 6:77 (1981), each of whichis incorporated herein by reference). Liposomes, for example, whichconsist of phospholipids or other lipids, are nontoxic, physiologicallyacceptable and metabolizable carriers that are relatively simple to makeand administer. “Stealth” liposomes (see, for example, U.S. Pat. Nos.5,882,679; 5,395,619; and 5,225,212, each of which is incorporatedherein by reference) are an example of such encapsulating materials, asare cationic liposomes, which can be modified with specific receptors orligands, for example, to target muscle tissue or brain (Morishita etal., J. Clin. Invest., 91:2580-2585 (1993), which is incorporated hereinby reference).

The route of administration of a composition containing an agent thatmodulates the sleep/wake cycle or circadian regulated locomotor activitywill depend, in part, on the chemical structure of the molecule.Polypeptides and polynucleotides, for example, are not particularlyuseful when administered orally because they can be degraded in thedigestive tract. However, methods for chemically modifying polypeptides,for example, to render them less susceptible to degradation byendogenous proteases or more absorbable through the alimentary tract arewell known (see, for example, Blondelle et al., supra, 1995; Ecker andCrook, supra, 1995). In addition, a polypeptide agent can be preparedusing D-amino acids, or can contain one or more domains based onpeptidomimetics, which are organic molecules that mimic the structure ofpeptide domain; or based on a peptoid such as a vinylogous peptoid.

A composition as disclosed herein can be administered to an individualby various routes, including, for example, topically, orally orparenterally, such as intravenously, intramuscularly, subdermally, orsubcutaneously, or by passive or facilitated absorption through the skinusing, for example, a skin patch or transdermal iontophoresis,respectively, or using a nasal spray or inhalant, in which case onecomponent of the composition is an appropriate propellant. Preferably,the composition is administered orally, for example, as a component of afood or beverage, or is administered in a time released formulation.Where a group of subjects is to be treated, for example, a herd of farmanimals, the composition can be incorporated in the livestock food orwater.

One skilled in the art would know that the amount of the composition toadminister to a subject depends on many factors including the age andgeneral health of the subject as well as the route of administration. Inview of these factors, the skilled artisan would adjust the particulardose as necessary. Appropriate amounts having the desired efficacy canbe determined using routine methods. When humans are to be treated, theformulation of the composition and the routes and frequency ofadministration are determined, initially, using Phase I and Phase IIclinical trials.

A composition for oral administration can be formulated, for example, asa tablet, or a solution or suspension form; or can comprise an admixturewith an organic or inorganic carrier or excipient suitable for enteralor parenteral applications, and can be compounded, for example, with theusual non-toxic, pharmaceutically acceptable carriers for tablets,pellets, capsules, suppositories, solutions, emulsions, suspensions, orother form suitable for use. The carriers, in addition to thosedescribed above, can include glucose, lactose, mannose, gum acacia,gelatin, mannitol, starch paste, magnesium trisilicate, talc, cornstarch, keratin, colloidal silica, potato starch, urea, medium chainlength triglycerides, dextrans, and other carriers suitable for use inmanufacturing preparations, in solid, semisolid, or liquid form. Inaddition auxiliary, stabilizing, thickening or coloring agents andperfumes can be used, for example a stabilizing dry agent such astriulose (see, for example, U.S. Pat. No. 5,314,695). Additionalformulations can be determined based on the particular subjects to betreated.

The following examples are intended to illustrate but not limit theinvention.

EXAMPLE 1 Circadian-Regulated Genes in Drosophila

This example provides a method for identifying the expression ofcircadian-regulated plant genes (see, also, Ceriani et al., supra,2002).

Circadian behaviors take place at regular intervals due to the action ofa cell autonomous clock, which marks time even in the absence ofenvironmental information. This molecular clock relies on negativefeedback loops, in which the positive driving forces control theexpression of the negative components, which, in turn, blocktranscription at their own promoters (Young and Kay, supra, 2001). Thisoscillation at the mRNA level is only the first step towards sustainablemolecular rhythms, which are accomplished by introducing a number of“delays” affecting message stability (Suri et al., EMBO J. 18:675,1999), protein stability (Kloss et al, Cell 94:97, 1998) or controllingsubcellular localization (Saez and Young, Neuron 17:911, 1996).

Although there is a relatively good understanding of how these molecularoscillations are generated, a clear link between the oscillator and thedownstream biological processes under clock control has not beenidentified. Previous attempts to describe the extent of circadiantranscriptional regulation identified a handful of clock-controlled“output” genes. A cDNA library screening identified about 20 oscillatingmRNAs, most of which have unknown functions (Van Gelder et al., Curr.Biol. 5:1424, 1995). Alternative approaches included the screening ofsubtractive cDNA libraries, which retrieved circadian regulated gene-I(Rouyer et al., EMBO J. 16:3944, 1997) and takeout (Sarov-Blat et al.,Cell 101:647, 2000 and differential display, which identified vrille(vri, (Blau and Young, Cell 99:661, 1999).

To carry out a more global examination of clock-controlledtranscription, high density oligonucleotide-based arrays were used. Thisapproach was successfully employed in Arabidopsis uncovering anastonishing range of physiological processes under circadian control(Harmer et al, supra, 2000).

Clock-Controlled Transcription at all Times

Steady-state RNA levels were followed using DNA GeneChip™ microarrays(Affymetrix) spanning the entire Drosophila genome. Temporal profilingof 13,500 probe sets was carried out with a 4 hr time resolution duringtwo consecutive days under “entrained” (light-dark cycles) and“free-running” (in constant darkness) conditions. Time courses for theDNA chipping experiments were carried out using Drosophila yw as wildtype control, and yw/clk^(jrk) as the mutant with impaired clockfunction. Newly eclosed wild type or mutant flies were entrained for 5days to a 12 hr:12 hr light:dark regime under constant temperature (25°C.). Time courses were performed either under light-dark (LD or“entrained”) or under constant dark (DD or “free-running”) conditions.Samples were collected every 4 hr for 2 consecutive days, andimmediately frozen in dry ice. Heads and bodies were kept apart forRNA/protein extraction.

For the DD time courses flies were entrained to LD cycles during 5 days,then released into constant darkness; samples were taken during thefirst and second days in DD. Total RNA was prepared from fly heads orbodies homogenized in TRIZOL reagent (Invitrogen Corp., CarlsbadCalif.). RNA was purified using RNeasy kit (Qiagen). 7.5 μg of total RNAwere hybridized to Drosophila GeneChip™ microarrays (Affymetrix). cDNAsynthesis, biotin-labeling of cRNA, and hybridization of the chips werecarried out as recommended by the manufacture. Data analyses wereperformed using Microarray Suite™ software (Affymetrix) and GeneSpring™software (Silicon Genetics) software packages. Each sample washybridized to two DNA GeneChip™ microarrays to test the reproducibilityof the technique (Harmer et al., supra, 2000). The mean hybridizationsignal strength and the standard error of the mean for each probe setwas calculated from the duplicate hybridizations.

To identify the cyclic mRNAs among the pool of expressed genes COSOPT,which is an analytical algorithm developed for detection and statisticalcharacterization of rhythmic gene expression in gene array experimentswas used. Briefly, data is fit to 100,000 cosine test waves and thesignificance of each fit is then determined empirically by temporallyrandomizing the data sets; those genes whose traces fit a cosine wavewith a period between 20 and 28 hours are scored as cycling with a givenprobability.

COSOPT imports data and calculates the mean expression intensity and itscorresponding standard deviation (SD). It then performs an arithmeticlinear-regression detrend of the original time series. The mean and SDof the detrended time series are then calculated. COSOPT does notstandardize the linear-regression detrended time series to standardnormal deviates, thus allowing COSOPT to quantitatively assessoscillatory amplitude much more directly. Variable-weighting ofindividual time points (as in SEMs from replicate measurements) can beaccommodated during analysis for the presence of rhythms in terms of auser-specified number and range of periods (test periods spaceduniformly in period-space).

Specifically, for each test period, 101 test cosine basis functions (ofunit amplitude) are considered, varying over a range of phase valuesfrom (plus one-half the period) to (minus one-half the period), i.e.,such that phase is considered in increments of 1% of each test period.COSOPT calculates, for each test cosine basis function, theleast-squares optimized linear correspondence between thelinear-regression-detrended data, ylr(x), and the test cosine basisfunction, yb(x), as a function of x, i.e., such that the approximationof ylr(x) by the test cosine basis function, yb(x), is optimized acrossall values, x, in terms of two parameters, ALPHA and BETA, wherebyylr(x)˜ALPHA+BETA*yb(x). The quality of optimization possible by thetest cosine basis function is quantitatively characterized by the sum ofsquared residuals between ylr(x) and the approximation given by{ALPHA+BETA*yb(x)} (referred to as CHI2, for Chi-squared).

The values of CHI2 are used to identify the phase at which the optimalcorrespondence between ylr(x) and yb(x) is obtained for each testperiod, i.e., the phase giving the smallest CHI2 value corresponds tothe optimal phase. Thus, for each test period are assessed these valuesof ALPHA, BETA, and CHI2 at the optimal phase. Interpretively, BETA nowrepresents an optimized, parametrized measure of the magnitude of theoscillatory amplitude expressed by ylr(x) in relation to, or as modeledby, a cosine wave of the corresponding period and optimal phase.Empirical resampling methods are employed to assess statisticalprobabilities of significance directly in terms of this parameter BETAat each test period and corresponding optimal phase, thus assessingstatistically the probability that a significant rhythm is present inylr(x) (in relation to or as modeled by a cosine functional form of thecorresponding period and optimal phase).

One thousand Monte Carlo cycles are carried out, in which surrogaterealizations of ylr(x) are generated by both (i) randomly shufflingtemporal sequence, and (ii) adding pseudo-Gaussian-distributed noise toeach surrogate point in proportion to the corresponding value of pointuncertainty (i.e., replicate SEM, for example). In this way,specifically accounted for in the surrogate realizations are both (i)the influence of temporal patterning, and (ii) the magnitude ofpoint-wise experimental uncertainty. Then, as with the original ylr(x)sequence, optimal values of ALPHA and BETA are determined, along with acorresponding CHI2, and retained in memory for each surrogate at eachtest period/optimal phase. For each test period/optimal phase are thencalculated the mean and standard deviation of the surrogate BETA values.These values, in relation to the BETA value obtained for the originalylr(x) series, are then used to calculate a one-sided significanceprobability based on a normality assumption, which, in fact, issatisfied by the distribution of BETA values obtained from the 1000randomized surrogates. A summary of the analytical session is thenproduced for each time series, composed of entries for only those testperiods that correspond to CHI2 minima.

In the L Dyw head time course, COSOPT scored 121 genes as cycling withp<0.01 (Table 1). This result likely is an underestimate of the totalnumber of cycling genes, as previously characterized clock componentssuch as per (which cycles with p=0.015) and clk (p=0.03) fall outside ofthis category. Since each individual cycling gene could not beconfirmed, the very conservative p value criterion was used to selectclock-controlled genes for further study.

TABLE 1 COSOPT assigned a p value to each putative cycling gene* Cyclinggenes p < 0.01 p < 0.025 p < 0.05 Heads 120 478 1206 Bodies 177 524 1144*see Young and Kay, supra, 2001.

Cycling genes were grouped in clusters according to the phase of peakexpression. The phase distribution is shown in Ceriani et al., supra,2002 (see FIG. 1A, demonstrating that output genes peak at differenttimes in fly heads and bodies. Genes were grouped according to the phasecalculated by COSOPT; each cluster represents genes peaking at thespecified time ±2 hr. ZT0 (“Zeitgeber Time 0”) refers to the time whenlights are switched on. Genes are expressed as percentage of the totalnumber of cycling ones.) Clock-controlled genes peak at all times duringthe day. More than 100 genes were identified by COSOPT as cycling with ap<0.01 in the head time course, and were plotted according to the peakof expression. A detailed list of cycling genes and corresponding phasescan be found in Table 2).

This approach allowed the identification of several novel phases of peakexpression (ZT4, ZT8, ZT12), and allowed a direct comparison between thedifferent genes within the same experiment. All phases were similarlyrepresented with the exception of ZT 8, where a larger proportion of thegenes appeared to reach maximum levels. This result is in contrast tothose reported for Arabidopsis (Harmer et al., supra, 2000) andcyanobacteria (Golden et al., Ann. Rev. Plant Physiol. Plant Mol. Biol.48:327, 1997). Internal validation of this approach towards theidentification of novel output genes came from the observation thatpreviously characterized cyclers such as per, tim, vri, to, and even lowamplitude genes such as clk and cry, displayed a circadian pattern ingene expression in the expression array experiment.

To confirm the circadian nature of the newly identified target genes, anindependent experiment under free-running conditions was performed. Oneof the hallmarks of clock-controlled activity is the persistence of therhythms in the absence of environmental cues. Accordingly, theproportion of the novel outputs that showed reliable cycling profilesunder constant darkness was determined. One of the limitations of thisanalysis under constant dark (DD) conditions is the “dampening” of thesignal amplitude, which partially results from the de-synchronization ofindividual cells in the absence of resetting environmental cues (Hardin,Mol. Cell. Biol. 14:7211, 1994). The data from both experiments aresummarized in Table 2.

TABLE 2 Cycling genes in fly heads (p < 0.01) are categorized by knownor predicted function. Gene ID lists either the gene name (if known) orthe Celera Transcript (CT) number, as well as the associated p value andphase under entrained (LD) and free running (DD) conditions. LD DDexperiment experiment Gene ID Function p-Beta Phase p-Beta Phase CELLADHESION CT35785 8.27E−03 21.3 Trn tartan 7.68E−03 9.7 METABOLISM CT1116succinyl CoA transferase 4.64E−03 4.2 CT22171 isocitrate dehydrogenase(NADP+) 4.59E−03 20.6 2.35E−01 21.1 CT34968 3-OH isobutyratedehydrogenase 4.19E−03 3.2 3.07E−02 2.5 CT12987 short-branched chainacyl CoA dehydrogenase 4.77E−03 0.5 9.06E−02 21.1 CT41571 aldehydedehydrogenase 9.50E−03 18.5 8.40E−02 22.0 CT1187 choline phosphatecytidil transferase 9.92E−03 8.8 2.30E−02 8.8 CT11757 anon 23-Da4.04E−03 14.6 Hmgs hydroxymethylglutaryl-CoA synthase 6.87E−03 21.6CT16527 cholesterol O acyl transferase 8.02E−03 7.8 CT40163Angiotensin-converting enzyme 2.26E−03 13.4 CT12411 oxido reductase7.54E−03 8.4 DEVELOPMENT CT27880 yellow-d2 5.51E−03 8.8 4.96E−03 15.0Idgf1 Imaginal Disc Growth Factor 1 6.02E−03 20.7 mbc myoblast city5.04E−03 11.7 DETOXIFICATION cyp6a21 Cytochrome P450 6a21 3.84E−03 21.26.82E−03 2.2 Ugt35b UDP-glycosyltransferase 35b 4.04E−03 3.7 9.80E−033.0 CT38753 gluthathione S-transferase 4.95E−03 5.6 CT38747 gluthathioneS-transferase 7.51E−03 5.1 cyp4e3 Cytochrome P450 4e3 3.30E−03 9.06.41E−03 14.2 cyp6d5 Cytochrome P450 6d5 6.05E−03 21.6 cyp6a17Cytochrome P450 6a17 9.37E−03 2.9 CT16545 glutathione transferase2.92E−03 7.8 1.58E−01 2.9 cyp6a2 Cytochrome P450 6a2 3.06E−03 7.91.49E−02 13.0 cyp18 Cytochrome P450 18 6.87E−03 17.1 6.86E−03 15.7 HEMEMETABOLISM Alas 5-Aminolevulinate synthase 9.99E−03 22.5 CT34507 hemeoxygenase-like 8.79E−03 9.1 1.28E−02 14.2 LIGAND BINDING/CARRIER CT6764glucan alfa 1,4 glucosidase 4.20E−03 18.4 9.98E−02 22.5 glob1 globin 16.68E−03 20.1 1.22E−01 21.2 CT33362 7.58E−03 20.4 2.36E−02 22.0 CT284952.79E−03 13.9 2.94E−02 21.9 NEUROTRANSMISSION ple tyrosine3-monooxygenase 4.52E−03 2.4 6.62E−03 1.6 Eaat2 Excitatory amino acidtransporter 2 4.36E−03 13.2 Dat Dopamine N acetyltransferase 5.18E−039.5 CT42497 glycine transporter 5.64E−03 6.9 5.77E−03 13.1 b

black, glutamate decarboxylase 7.66E−03 16.2 PHOTORECEPTOR CryCryptochrome 3.65E−03 1.1 PROTEASE INHIBITORS serpin serine proteaseinhibitor 8.34E−03 20.0 CT37177 9.29E−03 14.6 CT37195 8.24E−03 10.4PROTEIN SYNTHESIS/DEGRADATION CT18196 ubiquitin thiolesterase 2.23E−0313.1 7.31E−04 16.5 pros26 proteasome 26 kDa protein 4.89E−03 8.71.69E−02 8.4 pros26.4 Proteasome 26S subunit subunit 4 ATPase 7.14E−037.9 CT28749 26 S proteasome regulatory subunit p39A 6.22E−03 8.91.44E−02 10.7 CT7240 translation initiation factor (elF-5) 7.44E−03 8.52.91E−02 9.5 CT39962 tRNA synthase 6.46E−03 20.8 PROTEIN FOLDING CT16169heat shock protein 27-like 7.44E−03 21.9 Cnx99A Calnexin 99A 2.81E−0317.4 6.61E−02 19.2 TRANSCRIPTION CT30663 6.96E−03 11.6 tim Timeless4.66E−03 16.4 4.58E−03 17.4 Mad Mothers against dpp 4.97E−03 14.5 vriVrille 5.47E−03 13.6 1.58E−03 15.4 CT31519 5.08E−03 2.8 3.43E−01 7.5CT19628 6.28E−03 16.6 CT17296 4.78E−03 14.1 1.11E−02 17.4 CT18631 TfllA-L 8.42E−03 9.3 140up upstream of Rpll14 9.13E−03 17.5 9.15E−02 15.4SIGNAL TRANSDUCTION Akap200 protein kinase A anchoring protein 6.23E−0315.6 Pk61C Protein kinase 61C 2.92E−03 10.8 2.32E−02 14.1 Pka-C3 ser/thrkinase 4.85E−03 15.3 5.66E−02 7.6 CT35755 9.09E−03 21.5 1.93E−01 4.4CT27512 Ras small monomeric GTPase 8.14E−03 9.3 1.16E−03 12.4 SlobSlowpoke binding protein 3.04E−03 18.0 2.21E−03 16.3 CT34849 Chd649.76E−03 5.5 STRESS RESPONSE CT25938 superoxide dismutase (Cu—Zn)3.26E−03 8.6 Hsp22 Heat shock protein 22 7.82E−03 17.6 to Takeout8.26E−03 21.7 5.34E−02 2.1 STRUCTURAL PROTEINS dlp heparin sulfateproteoglycan 7.39E−03 11.2 Sulf1 N-acetylglucosamine-6-sultase 8.98E−038.8 1.26E−02 9.4 betaTub56D betaTubulin56D 8.71E−03 10.0 1.45E−01 22.9CT31310 8.92E−03 6.2 CT31613 Thrombospondin 2.25E−03 10.3 2.38E−02 11.9CT15377 9.13E−03 5.2 2.29E−02 3.0 Cpn Calphotin 8.94E−03 7.2 2.98E−029.9 capu cappuccino 9.98E−03 18.5 TRANSPORTERS CT42567 high affinityinorganic phosphate:sodium transporter 3.83E−03 0.2 4.57E−02 22.9CT30701 glucose transporter 8.23E−03 7.7 trpl trp-like 6.66E−03 9.8CT16777 8.63E−03 8.6 1.31E−02 13.9 UNKNOWN FUNCTION CT12588 8.91E−0323.5 2.87E−02 17.9 CT31141 9.90E−03 15.2 CT28701 7.65E−03 8.3 CT346318.27E−03 8.9 CT6802 7.37E−03 9.4 CT32204 3.85E−03 7.1 2.38E−03 7.7CT16557 9.34E−03 8.8 2.14E−03 16.2 CT16503 7.86E−03 2.2 7.18E−03 4.9CT33900 7.94E−03 18.9 CT22515 9.90E−03 14.1 CT33647 4.06E−03 1.07.49E−03 19.6 CT18564 9.89E−03 21.8 CT25838 5.23E−03 8.7 CT302568.04E−03 20.5 CT33484 3.57E−03 18.4 6.09E−03 21.0 CT32008 6.86E−03 8.3CT28709 8.73E−03 7.9 CT31865 2.26E−03 9.2 1.23E−03 14.3 CT32600 7.94E−0322.7 1.01E−01 22.0 CT15908 4.83E−03 8.8 2.40E−03 13.2 CT32262 2.32E−038.3 1.02E−03 10.2 CT34135 9.46E−03 5.5 7.15E−02 16.3 CT34439 9.64E−033.3 1.66E−01 15.7 CT10556 4.40E−03 8.7 CT42539 5.48E−03 8.5 CT359169.64E−03 5.9 1.14E−02 7.5 CT33073 4.59E−03 7.2 3.78E−03 10.0 CT370442.37E−03 20.6 6.98E−03 0.9 CT29508 8.79E−03 19.1 1.41E−01 1.3 CT296122.71E−03 19.5 3.37E−03 19.3 CT35582 2.66E−03 8.5 1.03E−03 14.4 CT325968.92E−03 10.8 CT26517 9.97E−03 4.8 3.09E−01 17.4 CT29500 4.71E−03 21.41.85E−02 2.3 CT16351 4.78E−03 15.5 CT24597 9.47E−03 4.5 2.78E−01 16.1CT26954 9.35E−03 8.7 CT25972 8.46E−03 15.9

indicates data missing or illegible when filed

Circadian Transcriptional Regulation of Physiology

Although cyclic transcription is a signature of clock activity,important levels of regulation take place downstream. Several genesinvolved in various aspects of protein regulation were under rhythmictranscriptional control. Three genes were identified that cycle with apeak at ZT8 and are part of the 26S proteasome complex (see Ceriani etal., supra, 2002; FIG. 3A). pros26 encodes a multicatalyticendopeptidase that is part of the central 20S barrel-shaped structure(Saville and Belote, Proc. Natl. Acad. Sci. USA 90:8842, 1993). pros26.4and rpn9 are part of the 19S regulatory complex, and correspond to thesubunit 4 of the AAA-ATPase and the homolog of yeast rpn 9, which isrequired for assembly and stability of the proteasome, respectively. InDrosophila, different proteasome subunits are expressed throughoutdevelopment, possibly to control specific processes such as celldivision or morphogenesis (Haass and Kloetzel, Exp. Cell Res. 180:243,1989). A putative de-ubiquitinating enzyme, the homolog of the mouseUBPY, with peak expression at ZT12 (see Ceriani et al., supra, 2002;FIG. 3A), also was identified. This cyclic pattern was confirmedindependently by northern blot analysis. Northern blot analysis ofindependent head and body time courses confirmed the cyclic nature ofthe candidates tested and the timing of mRNA peaks (Suri et al., EMBO J.18:675, 1999). “Gene ID”, which refers to the cDNA used to probe thedifferent time courses, had an expected peak time predicted by the arrayexperiment, as follows: Gene ID CT 38753, ZT4; CT 8171, ZT4/8; CT 18196,ZT12; CT 33647, ZT20; CT 33647, ZT0; and CT12127, ZT8. De-ubiquitinatingenzymes remove the polyubiquitin chain from conjugated proteins prior totheir degradation by the proteasome. These enzymes can regulatedegradation by the proteasome, or can be involved in ubiquitinationprocesses not directing protein degradation, but rather subcellularlocalization. These results indicate that temporal regulation of theproteosome can be important in Drosophila physiology.

The observation that the expression of both the rate limiting enzyme onheme biosynthesis, d-aminolevulinate synthase (alas), and the ratelimiting enzyme of heme degradation, heme oxygenase, cycle in Drosophilaheads indicates that heme metabolism is tightly regulated by the clock(with a peak at ZT 20 and ZT8, respectively; see Ceriani et al., supra,2002; FIG. 3B). Heme is involved in respiration, oxygen transport,detoxification, and signal transduction processes. However, as achelator of iron, heme may promote deleterious cellular effects such asoxidative membrane damage. Thus, maintaining a proper balance betweenheme biosynthetic and degradation pathways is crucial for cellularhomeostasis (Ryter and Tyrrell, Free Rad. Biol. Med, 28:289, 2000).

In insects, P450 enzymes are thought to be involved in the biosyntheticpathways of ecdysteroids and juvenile hormones, and, as such, play arole in insect growth, development, and reproduction, as well asmetabolize natural plant products and insecticides, resulting inbioactivation or detoxification (Feyereisen, Ann. Rev. Entomol. 44:507,1999). The present study revealed 6 different cytochrome P450 genes,cyp4e3, cyp6a2, cyp6a17, cyp6a21, cyp6d5, and cyp18 cycle with differentphases (ZT 8, 4, 0, 20, and 16, respectively; see Ceriani et al., supra,2002; FIG. 3C). The only functionally characterized enzyme thus far iscyp6a2, which is involved in the metabolism of organophosphorusinsecticides. Detoxification often occurs in two phases. The initialcompound can be transformed into a more reactive species (usually viaredox reactions catalyzed by cytochrome P450 enzymes); in the secondphase, highly polar groups such as UDP-glucuronosyl or glutathione areadded either to the products of the first phase or, in some cases,directly to the toxic chemicals. The enzymes involved in this secondstage phase include UDP-glucuronosyl transferases (ugt) and gluthathioneS-transferases (GST's). Products of phase II are highly hydrophilic, canno longer cross membranes, and are eliminated by secretion. The presentstudy revealed that ugt35b and several GST's also cycle in fly heads(see Ceriani et al., supra, 2002; FIG. 3D), indicating that multiplesteps in the biotransformation process are under circadian control.

Another gene that was under circadian control is pie, indicating thatneurotransmission is a clock-controlled process. ple codes for thetyrosine 3-monooxygenase (also known as tyrosine hydroxylase), the firstand rate limiting enzyme in dopamine biosynthesis. Dopamine is anintermediate in cuticular esclerotization and also functions as asignaling molecule in the fly nervous system. Dopamine can modulatecertain forms of learning such as female sexual receptivity andhabituation, as well as motor neuron activity and neuromuscular functionin larva (Neckameyer, Learn. Mem. 5:157, 1998; Cooper and Neckameyer,Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 122:199, 1999). Thepresent studies revealed that ple is expressed at very low levels in flyheads, but cycles with high amplitude, peaking at ZT 4 under bothentrained and free running conditions. ple expression falls below thelevel of detection in clk^(jrk) (a mutation that impairs clock function;Allada et al., Cell 93:791, 1998). This result indicates that ple is adirect CLOCK target. ple represents one of the several examples ofhomolog genes cycling in both flies and mammals (Carre and Kay, PlantGene Research: Basic Knowledge and Application (Dennis et al, Eds.,1995)).

The Clock Controls Different Sets of Genes in Different Tissues

To provide a more comprehensive view of clock control in a wholeorganism, cyclic gene expression was analyzed using male bodies as thesource of total RNA, and compared these transcripts with those derivedfrom heads. COSOPT identified 177 genes that cycled with a periodbetween 20 and 28 hr (p<0.01; Table 3). As in the mouse, only a smallproportion of cycling transcripts (12 genes) overlapped between the twotissues, including some previously identified clock components (such astim, vri and cry). The analysis of the remaining genes revealed that alarge number of genes that cycle solely in fly heads, nevertheless areexpressed at medium to high levels in the bodies. The clock controlsdifferent subsets of genes in heads and bodies, with distributionsobserved of genes cycling in both tissues, cycling and expressed in onlyone, and expressed to mid-high levels in both but cycling in one. Thisresult indicates that differential transcriptional regulation acts inthis subset of clock outputs, in agreement to what has been found in themouse (Carre and Kay, supra, 1995).

Potassium Channel as an Output of the Clock Involved in SustainedRhythmic Behavior

The identification of the clock-controlled genes allowed aninvestigation to identify those genes crucial for the control ofrhythmic behavior. Among the candidates, slob (slowpoke binding protein)was identified. SLOB binds to the Ca2+-dependent voltage-gated potassiumchannel slowpoke (slo; Schopperle et al., supra, 1998), which, whenmutated causes behavioral defects (Atkinson et al, J. Neurosci. 20:2988,2000) and an altered mating song, also a hallmark of certain clockcomponents (Peixoto and Hall, Genetics 148:827, 1998). SLOB has beenshown to modulate SLO activity through the formation of a complex with a14-3-3 protein, that is a downstream of several signaling pathways (Zhouet al., Neuron 22:809, 1999). In flight muscle, slowpoke functions inaction potential repolarization (Elkins et al., Proc. Natl. Acad. Sci.USA 83:8415, 1986) and also was proposed to participate in therepolarization at the nerve terminal of the motoneurons (Gho andMallart, Pflugers Arch. 407:526, 1986).

slob mRNA cycled robustly in fly heads in LD and DD (Table II; seeCeriani et al., supra, 2002; FIGS. 4A (entrained conditions) and 4B(free-running conditions)). This pattern is lost in the clk^(jrk) mutantbackground, although it likely is not a direct CLK target because theoverall levels of expression remain unchanged. In addition, slo mRNAoscillated in phase with slob in LD and DD; the level of expression wasnear the limit of detection of the technique (see Ceriani et al., supra,2002; FIGS. 4A and 4B); slo cycling also was obliterated in theclk^(jrk) mutant.

TABLE 3 List of cycling genes in fly bodies (p < 0.01), as in

 table II. LD experiment Gene ID Function p-Beta Phase DEFENSE/IMMUNITYCT8705 peptidoglycan recognition protein 1.87E−03 22.20 Phas1eukaryotic-initiation-factor-4E 9.76E−03 23.00 binding protein Ag5rAntigen 5-related 2.10E−03 21.31 Chit chitinase-like 6.84E−03 7.60CT5624 chitinase-like 3.01E−03 17.25 CT30310 9.84E−03 15.12 CT291023.51E−03 5.40 CT16885 PGRP-SC1b 1.46E−03 8.50 DEVELOPMENT Idgf4 ImaginalDisc Growth Factor 4 2.20E−03 3.20 CT13185 dorso-ventral patterning inoogenesis 8.53E−03 4.90 DETOXIFICATION cyp9b2 4.91E−03 4.70 CT20826cyp18a1 4.07E−03 19.34 Cyp6gl 1.86E−03 8.03 CT38747 glutathionetransferase 2.29E−03 5.90 GstD1 glutathione S transferase B1 5.67E−037.30 CT35150 glutathione S-transferase-like 4.46E−03 7.50 CT35071UDP-glucuronosyltransferase 9.59E−03 4.90 ELECTRON TRANSFER Cyt-A5Cytochrome A5-related 7.50E−03 7.90 LIGAND BINDING/CARRIER CT3751triglyceride binding 6.09E−03 6.50 Mp20 Muscle protein 2 8.59E−03 18.85Dbi Diazepam-binding inhibitor 7.95E−03 8.60 PebIII ejaculatory bulbprotein III 4.14E−03 19.62 CT33980 neural Lazarillo 9.36E−03 19.72CT31326 antennal binding protein X-like 6.24E−03 3.90 CT32778 Odorantbinding protein 2.78E−03 5.40 CT21061 fatty acid binding protein-like1.94E−03 9.00 METABOLISM CT41369 2.57E−03 4.70 Wun phosphatidatephosphatase 6.99E−03 19.01 LysX Lysozyme X 9.50E−03 17.55 CT161873-hydroxyisobutyryl-CoA hydrolase 6.41E−03 0.50 CT24308 threoninedehydratase 6.48E−03 7.90 CT6492 maltase L-like 2.28E−03 6.80 Hmgshydroxymethylglutaryl-CoA synthase 6.40E−03 23.06 LvpH larval visceralprotein H 4.80E−03 7.50 Pepck Phosphoenolpyruvate carboxykinase 6.50E−0311.00 Scu 3-hydroxyacyl-CoA dehydrogenase 9.57E−03 8.90 CT17038 malatedehydrogenase 3.07E−03 8.10 CT36683 alpha-Amylase-like 1.74E−03 8.40CT6532 maltase H-like 4.49E−03 7.30 CT22063 triacylglycerol lipase-like9.88E−03 7.60 CT22069 hexokinase 2.84E−03 8.00 CT21171 carbonatedehydratase-like 6.81E−03 4.70 CT12913 ubiquinone biosynthesis 6.51E−0321.39 CT34308 UDP-glucose epimerase 5.17E−03 8.50 CT18319 pyrroline5-carboxylate reductase-like 6.39E−03 7.30 CT33098 alpha-glucosidase II3.93E−03 7.40 CT19053 carbonate dehydratase-like 5.82E−03 16.25 CT28913glucosylceramidase-like 7.84E−03 9.45 RfaBp retinoid- and fatty-acid2.28E−03 8.40 binding protein CT26924 glucose dehydrogenase-like6.49E−03 7.50 alpha-Est5 carboxylesterase 8.47E−03 10.00 RfaBp retinoid-and fatty-acid 1.50E−03 9.60 binding protein Pugformate--tetrahydrofolate ligase 8.23E−03 7.60 Lectin-galC1Galactose-specific C-type lectin 3.60E−03 9.00 alpha-Est7alpha-Esterase-7 9.35E−03 9.70 ade5 phosphoribosylaminoimidazole3.38E−03 9.50 carboxylase; EC: 4.1.1.21 Pdh photoreceptor dehydrogenase9.23E−03 6.90 CT30991 IMP cyclohydrolase 4.38E−03 9.80 CT24026alpha-galactosidase 7.24E−03 8.90 CT9666 transaldolase 3.14E−03 8.50CT39259 oxidoreductase 3.46E−03 3.10 CT41283 cholinephosphatecytidylyltransferase 6.56E−03 0.42 RfaBp retinoid- and fatty-acid1.50E−03 9.60 binding protein Pug formate--tetrahydrofolate ligase8.23E−03 7.60 Lectin-galC1 Galactose-specific C-type lectin 3.60E−039.00 CT33239 N-acetyl transferase 9.11E−03 15.44 CT31087 rRNAmethyltransferase 4.32E−03 13.20 CT34977 epoxide hydrolase 5.71E−03 6.30MUSCLE CONTRACTION Mhc myosin II heavy chain 6.81E−03 17.56 TpnC73FTroponin C at 73F 6.66E−03 17.79 PROTEASES CT15463 zinc carboxypeptidase7.73E−03 21.92 CT14806 vitellogenic carboxypeptidase 7.54E−03 6.80CT14378 carboxypeptidase-like (inactive) 3.89E−03 1.50 CT4209chymotrypsin 3.30E−03 22.58 CT37183 serine protease inhibitor 1.66E−039.00 CT25448 chymotrypsin 8.12E−03 21.11 CT8699 serine protease-like7.10E−03 22.52 CT19724 serine protease 6.97E−03 20.04 CT37173 serineprotease-like 1.98E−03 14.30 CT3996 cathepsin 4.41E−03 9.30 CT20780cathepsin L 5.44E−03 13.40 Ser99Da Serine protease 1 9.03E−03 23.59Ser99Db Serine protease 2 9.91E−03 23.18 CT29408 serine protease4.45E−03 22.77 gammaTry gammaTrypsin 2.77E−03 20.66 PROTEINSYNTHESIS/FOLDING CT25442 ribosomal protein K11-like 7.71E−03 20.20CT17486 chaperone 2.69E−03 19.57 SIGNAL TRANSDUCTION Pp1-13C ser/thrphosphatase 9.91E−03 5.80 CkIIbeta casein kinase II beta subunit7.15E−03 16.77 Cry cryptochrome 5.77E−03 4.10 Lk6 heat shock constructof Kidd 4.74E−03 5.20 CT22109 Accessory gland peptide 36DE 9.84E−0317.93 STRESS RESPONSE Cat Catalase 1.53E−03 8.80 CT33074 takeout4.71E−03 0.65 CT9894 heat shock protein 70 6.26E−03 23.78 STRUCTURALPROTEINS Gel Gelsolin 6.95E−03 7.80 Msp-300 Muscle-specific protein 38.02E−03 16.42 CT33880 cuticle protein-like 9.68E−03 17.33 Glt Glutactin4.72E−03 9.10 CT41348 A-band-protein-225 6.88E−03 17.09 TRANSCRIPTIONCT15944 7.39E−03 17.21 CT6009 9.47E−03 13.50 Tim timeless 3.98E−03 17.53TRANSPORTER Vha13 hydrogen-transporting two-sector 6.26E−03 21.66 ATPaseBY8473 ATP-binding cassette transporter 1.48E−03 22.77 CT27832sodium/phosphate cotransporter 4.17E−03 17.16 CT13124 zetaCOP 5.41E−0320.56 CT14766 ATP-binding cassette transporter 9.08E−03 18.53 CT30183sugar transporter-like 6.99E−03 8.00 CT10168 sodium/phosphatecotransporter 6.97E−03 18.12 CT19169 sugar transporter 6.06E−03 6.70CT33284 4.70E−03 8.90 CT15971 amino-acid permease-like 9.52E−03 17.98UNKNOWN FUNCTION CT19307 7.73E−03 20.48 CT21613 8.81E−03 17.97 CT358649.38E−03 14.41 CT14230 6.06E−03 6.50 CT14296 2.49E−03 6.60 CT238982.59E−03 9.30 CT23894 8.82E−04 7.50 CT21019 7.01E−03 5.70 CT90938.76E−03 18.52 Mst57Dc Male-specific RNA 57Dc 6.37E−03 16.93 GipGip-like 8.12E−03 9.80 CT35241 6.39E−03 9.30 CT25774 7.17E−03 8.00CT27394 3.05E−03 9.00 CT35953 8.23E−03 3.00 CT34370 9.41E−03 17.70CT22505 6.20E−03 7.60 CT12127 1.59E−03 7.70 CT35796 5.00E−03 17.12CT26922 4.52E−03 8.90 CT33647 3.27E−03 0.00 CT27834 2.31E−03 7.80CT18564 2.24E−03 23.50 CT35055 8.36E−03 7.50 CT35089 9.38E−03 5.00CT8227 5.83E−03 20.29 CT18118 9.50E−03 20.02 CT42569 1.54E−03 22.14CT40493 9.81E−03 9.00 CT33419 9.49E−03 1.95 CT34875 9.46E−03 15.36CT32604 9.65E−03 19.09 CT39634 6.22E−03 8.40 CT22395 5.73E−03 21.98CT33153 8.49E−03 5.20 CT12789 5.68E−03 21.28 CT1789 6.56E−03 2.80CT40966 7.27E−03 6.00 CT36877 6.76E−03 23.39 CT26804 3.60E−03 21.31CT26802 4.05E−03 21.61 CT28427 2.07E−03 5.40 CT38785 9.84E−03 2.50CT34717 4.17E−03 5.70 CT18134 3.68E−03 9.90 CT33073 5.64E−03 13.90CT37044 4.08E−03 3.30 CT33083 8.21E−03 9.30 CT29612 9.10E−03 20.14CT20261 4.25E−03 7.80 CT34939 1.41E−03 9.40 CT34936 8.21E−03 11.00CT37419 8.18E−03 13.90 CT36793 3.98E−03 16.56 CT18390 3.53E−03 1.30CT9987 9.65E−03 7.70 CT29500 4.76E−03 21.63 CT26166 kisir 4.69E−03 21.50CT35842 6.17E−03 8.60 CT32316 7.80E−03 3.40 CT16297 9.32E−03 17.56CT21565 9.20E−03 18.83 CT42272 5.07E−03 6.20 CT39472 Peritrophin-15a4.86E−03 20.12 CT22723 Senescence marker protein-3 3.39E−03 8.90

indicates data missing or illegible when filed

To validate the cycling of slo at the protein level, SLO proteinabundance was determined in light/dark cycles in fly head extracts bywestern blot analysis. Time courses were performed as described above.About 10 μl of wild type and mutant fly heads were loaded into precast4-15% acrylamide gels (BioRad). Gels were transferred in 15% methanolTRIS-glycine buffer onto MSI supported nitrocellulose. Primaryantibodies used were rabbit IgG anti-SLO (1:500) and mouse monoclonalIgG anti-hsp70 (1:2000, Sigma). Washes were performed in TBS (for SLO)or TBS 0.5% TWEEN detergent (for hsp70). Appropriate secondaryantibodies were detected using ECF (Amersham). The results revealed thatSLO oscillated with a peak at ZT20 (Ceriani et al., supra, 2002; FIG.4C).

In Drosophila, a considerable body of work identified a small group ofcells called ventral lateral neurons (LNv) as the pacemaker neurons. TheLNv's play a major role in the control of circadian locomotor activityunder entrained and free running conditions (Helfrich-Förster, J. Comp.Physiol. (A) 182:435, 1998). Although slo expression pattern has beenstudied extensively (Becker et al., J. Neurosci. 15:6250, 1995), littleis known about its expression in the regions of the brain relevant tocircadian control of behavior. To further characterize the role ofslowpoke as an output of the clock, SLO spatial distribution wasexamined in the fly head. Anti-SLO histochemistry was performed usingCNS whole mounts. Wild-type (Canton-S) flies were subjected to at least3 cycles of 12 hr: 12 hr light:dark. Males were sacrificed between ZT18-20 and their brains dissected in 1×PBS. The specimens were fixed in4% paraformaldehyde for 30-60 min at room temperature, with gentleshaking. Tissues were washed 3 times with 0.2 M phosphate; then washed 3further times in TNT (0.1M Tris HCl/0.3M NaCl (pH 7.4), 0.5% TRITONX-100 detergent); the duration of each wash was 20 min. The washedspecimens were preincubated in a blocking solution, composed of 4%normal donkey serum (diluted in TNT) for 2-3 hr.

Anti-SLO antibody was applied at a dilution of 1:100 directly in theblocking solution and incubated overnight at 10-12° C. with gentleshaking. The tissues were then washed 6 times (20 min each) in TNT andsubsequently incubated with Cy TM 5-conjugated secondary anti-rabbit IgGat dilution of 1:200 (Jackson ImmunoResearch; West Grove Pa.) for 2 hrat room temperature. All further steps were performed in the dark:Tissues were washed 3 times in TNT, followed by 3 times in 0.1Mphosphate buffer (6 washes; 20 min each). Brains were then mounted with2% n-propyl gallate (80% glycerol in 0.2 M phosphate buffer, pH 7.4).Samples were observed using an MRC600 laser-scanning confocal microscope(BioRad, Richmond, Calif.).

Immunocytochemistry analysis on whole mount brain preparations indicatedthat this channel is expressed in a subset of the LNv. Images wereproduced by collecting numerous Z series sections (approx. 2 μm each)then projecting them into one figure, which represents approximately 40μm of confocal stacking. Eighteen brains were processed in this manner;and in 16 of the specimens, prominent signals appeared in certainbilaterally symmetrical cell clusters which are located near theanterior rim of the medulla optic lobe. These cells appeared to beeither a subset of the clock-gene expressing Lateral Neurons (LNs) ornear their locations. SLO immunoreactivity within these putative LNsappeared to be cytoplasmic. Additional anti-SLO-mediated staining wasdetected less frequently. Signals were detected near the Kenyon cells ofthe mushroom bodies (in a medial region of the dorsal brain), which wereweak compared with the putative LN staining and observed in only 50% ofthe brains. In the majority of the specimens, relatively strong signalsappeared at the edges of the optic lobes, and within those gangliascattered, weak signals were erratically observed.

Prompted by evidence that SLO was involved in mediating locomotoractivity, the locomotor activity in two slo mutants, slo1 and slo4, wasexamined. slo1 was generated by EMS mutagenesis and slo4 by gammairradiation; the latter is a result of an inversion that deletes thepromoters required for specific expression in neural and muscle tissue.Wild type flies showed increased locomotor activity near dawn and dusk,and remained quiescent the rest of the day (see Ceriani et al., supra,2002; FIGS. 5 and 6; see, also, Hamblen-Coyle et al., J. Insect Behav.5:417, 1992). Furthermore, the bursts of activity did not merely followthe next environmental transition, but, instead, anticipated thetransition. These so called “startle effects” remained in some of themutants affecting core clock components when the flies were kept underentrained conditions (such as per⁰), making them appear rhythmic.Interestingly, norpA/per⁰ double mutants also displayed startle effects,indicating that this effect is not mediated by light through the visualpathway (Wheeler et al., J. Biol. Rhythms 8:67, 1993).

FIG. 5 of Ceriani et al. (supra, 2002) shows representative individualactograms of wild type CS and yw flies (left), and the mutants slo1,slo4, per⁰ and clk^(jrk). Flies were entrained for 5 days before theonset of the experiment. During the experiments flies were kept in LDfor 3-4 days, then switched to DD and monitored for at least anotherweek. Rhythmicity and total activity in LD and DD conditions wasdetermined using the Clocklab™ software package. Newly eclosed flieswere entrained to 12 hr:12 hr LD cycles for 3 days, and adult males wereplaced in glass tubes and monitored for activity with infrared detectorsand a computerized data collection system (Trikinetics, Mass.). Activitywas monitored in LD conditions for 3 to 4 days, when the flies werereleased into constant darkness (DD) at least for a week. Data wasanalyzed using Clocklab/Matlab software package. Only those flies thatwere alive 2 days after the analysis ended were taken into account.Periodogram analysis of flies that were scored as arrhythmic in Table IVproduced no strong peak that was statistically significant with p<0.001.Overall activity was calculated by averaging equivalent bins during LDor DD cycles for each fly, then taking an average of all the flieswithin each genotype.

Wild type CS and yw were rhythmic in LD and in DD, where the endogenousperiod became apparent (see Ceriani et al., supra, 2002; FIG. 5; Table4). per⁰ and clk^(jrk) mutants, which have defects in core clockcomponents, behaved differently under entrained conditions; per⁰ fliesremained mostly rhythmic in LD, whereas clk^(jrk) often were not (seeabove). slo1 and slo4 mutants were arrhythmic in LD, although thestrength of the phenotype varied with the mutation. The lack ofrhythmicity persisted under free running conditions (Ceriani et al.,supra, 2002; FIG. 5; Table IV).

TABLE 4 A null mutation in the slowpoke potassium channel does notchange the overall activity levels Rhythmic lines Activity (%) (totalcounts/day) Genotype (n) LD DD LD DD CS (53) 100 100 1017 1204 yw (74)86 88 611 910 slo4 (68) 1 1 810 813 slo1 (41) 29 54 808 820 clk^(jrk)(41) 22 7 1110 1518 per^(o) (35) 71 3 695 1141 Number of flies analyzedwithin each genotype is expressed in parentheses.

Several scenarios can account for these observations. A mutation inslowpoke can cause arrhythmicity if it directly affects the outputpathway controlling behavior, i.e., affecting the excitability of themotor neurons that control behavior. Alternatively, the mutation can actat the level of the pacemaker neurons by reducing the coupling betweenthe LNs, which also can cause the observed lack of behavioralrhythmicity. Slowpoke also can “gate” (McWatters et al., Nature 408:716,2000) fly locomotor activity that would be regulated by additionalunidentified components.

To determine if this mutation caused general sluggishness, which couldper se result in arrhythmicity, the total activity displayed by thedifferent genotypes under LD and DD conditions was quantified. Wild typeflies were slightly more active under constant darkness, but both slomutants responded the same in both environmental conditions. Moreimportantly, the overall levels of activity were not different fromthose of the wild type flies (Table 4). When the actograms of wild typeand slo4 mutant flies were superimposed, these average activity plotsrevealed features that were not apparent when inspecting individualflies. The most striking difference was the lack of anticipation andresponse to the transitions (startle effect) in the slo4 flies (seeCeriani et al., supra, 2002; FIG. 6, showing average activity plots forwild type and slo4 mutant flies; activity records of the LD portion ofthe experiment for 53 wild type flies and 28 slo4 flies were used forthe analysis; to superimpose the separate animal records the levels ofactivity were normalized per fly per day). This result indicates thatthe gating that consolidates behavior around dawn and dusk was missingin the slo4 flies.

In summary, steady state mRNA levels were examined using high densityoligonucleotide arrays for circadian patterns of expression in the flyhead and body. This analysis identified several genes that are known tobe rhythmically expressed, as well as several hundred genes of known andunknown function that also are under clock control. A number of aspectsof fly physiology ranging from basic cellular metabolism toneurotransmission, stress resistance and detoxification were found to beunder control of the biological clock. As in the mouse, few genes cycledin both heads and bodies, indicating that tissue specificity is animportant component of circadian transcriptional regulation. Severalcycling genes in the fly also cycled in the mouse, suggesting these geneare important output mediators or core clock components. Behavioralfollow-up of one of genes, slo, indicates it is a central regulator oflocomotor activity. The notion that a potassium channel is involved inthe generation of rhythmic activity was proposed a number of years agofollowing the analysis of membrane conductance changes in isolatedretinal neurons of the mollusk Bulla (McMahon and Block, J. Comp.Physiol. A161:335, 1987; Michel et al., Science 259:239, 1993). Thisobservation, together with the finding that potassium currents arecircadian regulated in the mouse, and that expression of Kcnma1, theslowpoke mouse ortholog, cycles (see Example 2), indicates that thismechanism of control of rhythmic activity is evolutionarily conserved,and utilized in more complex organisms.

EXAMPLE 2 Mammalian Circadian-Regulated Genes

Coordinate gene regulation can function to organize specific biologicalprocesses in a developmental, spatial, or temporal manner. The lightdark cycle is an important temporal consideration as it influences manyphysiological processes in organisms ranging from cyanobacteria, fungi,plants, flies, mice, and humans (Young and Kay, Nat. Rev. Genet. 2:702,2001). This temporal regulation is accomplished by a circadian clock,which, in mammals, resides in the suprachiasmatic nucleus (SCN) of thehypothalamus. In mammals, many important functions are under circadiancontrol, including the sleep-wake cycle, hormonal rhythms, bodytemperature, and feeding. Powerful Drosophila and mammalian geneticshave implicated PAS (Per-Arnt-Sim) domain-containing transcriptionfactors in the regulation of clock gene expression (Dunlap, Cell 96:271,1999). Characterization of several of these factors has revealed afeedback inhibitory loop that governs transcriptional rhythmicity with aperiod of 24 hours (Young and Kay, supra, 2001; Dunlap, supra, 1999).Two positive activators, Clock and Mop3/Bmal1, regulate expression byinteracting with enhancer elements, termed E-boxes. Target genes ofthese activators include several repressors, including the Per proteins,Per1, Per2, and Per3, and the Cryptochrome molecules, Cry1 and Cry2,which function to inhibit the Clock/Mop3 complex generating a circadianoscillation of 24 hours (see Young and Kay, supra, 2001).

Despite this growing understanding of the mechanism of circadian generegulation, the link between transcriptional output and physiology undercircadian control has been lacking. To address this problem, highdensity oligonucleotide DNA arrays were used to identify mRNAtranscripts exhibiting circadian expression patterns in the SCN andliver of mice entrained to a 12 hr: 12 hr light:dark cycle andsubsequently placed in constant darkness (see, also, Panda et al.,supra, 2002b). The dataset obtained confirmed the expression of manygenes previously known to be circadian regulated, and identifiedadditional circadian regulated genes of known and unknown function inclock control. Analysis of orchestrated expression patterns of thesegenes revealed transcriptional networks that underlie temporalcoordination of physiological processes. A publicly accessible databasehas been constructed on the world wide web, at the URL“expression.gnf.org\circadian”, where users can query for circadianregulated genes in the SCN or liver, or for the temporal expressionpatterns for their genes of interest.

For these experiments, 7 to 8-week old male C57BL/6J mice were entrainedon a 12 hr: 12 hr light: dark cycle for two weeks, then placed inconstant darkness for one full day. Ten animals were sacrificed per timepoint starting at CT18 for two full days every 4 hr. Brains and liverswere rapidly dissected under dim red light (15 W Kodak safe lamp filter1A); the SCN were quickly dissected under bright light in a dissectingmicroscope and placed in a 5 μl drop of TRIZOL reagent (Invitrogen).Total RNA was prepared, and samples were labeled and hybridized induplicate (or more) to mouse (U74A) high-density oligonucleotide arrays(GeneChip® Murine Genome U74 Set Version 2; Affymetrix) as previouslydescribed (Lockhart et al., Nat. Biotechnol. 14:1675, 1996; Wodicka etal., Nat. Biotechnol. 15:1359, 1997; Sandberg et al., Proc. Natl. Acad.Sci. USA 97:11038, 2000, each of which is incorporated herein byreference). Primary image analysis of the arrays was performed usingGENECHIP 3.2 software (Affymetrix), and images were scaled to an averagehybridization intensity (average difference) of 200. The COSOPTalgorithm, which generates a p-value (pMMC-beta) and assigns phase, wasused to detect cycling genes (see Example 1). Transcripts having ap-value of 0.1 or less were considered cycling, and binned intocircadian times every four hours, from CT2 to CT22. Expression patternsfor these transcripts were visualized using GENESPRING software (SiliconGenetics; Redwood City Calif.).

The availability of a comprehensive circadian dataset from two differenttissues offered an opportunity for a systematic analysis oftranscriptional output of the clock. Approximately the same number(approx. 10%) of detectably expressed genes were under circadian controlin both SCN and liver. In addition, several genes previously identifiedas clock regulated such as Cry1, Per2, and Mop3 were detected in thedataset, while others such as Per1 apparently were cycling, but fellbelow the conservative detection threshold set for the present study.

The peak expression of the identified genes were distributed throughoutthe circadian cycle, with the most populous clusters in the SCN beingCT10 (10 hours after subjective dawn) and CT22, roughly anticipatingdusk and dawn, respectively (see Panda et al., supra, 2002b; FIGS. 1A to1C). 337 SCN and 335 liver transcripts were binned by COSOPT into 6circadian times (CT). In liver, the largest clusters of circadianregulated transcripts occurred at CT6 and CT14 (see Panda et al., supra,2002b; FIGS. 1A to 1C), likely reflecting the distinct temporalorganization of the different physiologies mediated by the liver (e.g.,metabolism). A comparison of overlap between cycling genes in the SCNand liver revealed only 28 genes in common (see Panda et al., supra,2002b; FIG. 1D), including the clock component, Per2, and several geneswhose transcripts were previously not known to cycle (see, also, Table5). Genes that cycle in all output tissues can represent basic cellularoutputs or critical components of the circadian clock (e.g., Per2). Mostgenes, including Per2, were delayed in peak expression in liver ascompared to the SCN, usually by about 4 hr to 8 hr. However, severalgenes, including tubulin-5 (Tubb5) and a transforming growthfactor-1-induced transcript (Tgfbli4), were coordinately regulated withthe same phase in both tissues.

To further investigate the overlap in cycling genes between the SCN andliver, the average level of expression was determined for each cyclinggene in both tissues. The distribution of liver cycling transcripts inSCN and liver by abundance was determined, and the relative abundance ofeach transcript (average AD value across time course) was obtained forliver cycling transcripts (pMMC-beta <0.1) from the liver and SCN.Similar results were obtained when comparing the relative abundance ofSCN cycling transcripts in SCN and liver. This analysis revealed that asignificant number of cycling genes in the liver do not cycle in the SCNbecause they are not detectably expressed there. Murinoglobulin 2(mug2), is an example of such a gene, whose expression is tightlyrestricted to (and circadian regulated in) the liver (see Panda et al.,supra, 2002b; FIG. 1I and Suppl. Figure S2; see, also, Lorent et al.,Differentiation 55:213, 1994). This simple mechanism, however, fails toexplain the fact that many genes with detectable and equivalent levelsof expression in both tissues cycle only in one of the two tissues. Forexample, carbon catabolite repression 4 homolog (Ccr4) is expressed atapproximately the same levels in the dataset in both SCN and liver, yetonly cycles in the liver (see Panda et al., supra, 2002b; FIG. 1I andSuppl. Figure S2). The previously described circadian pattern ofexpression of Ccr4 in several other peripheral tissues, includingkidney, heart, spleen, and retina, highlights the tissue specificcomponent to circadian transcriptional rhythmicity in mammals (Wang, etal., BMC Dev. Biol. 1:9, 2001). These results indicate that asignificant percentage of the genome (approximately 10%) is under tissuespecific circadian regulation, and underscore the importance of temporalorganization for many physiological processes.

TABLE 5 SCN Liver Cosopt.pMMC- Cosopt.pMMC- Probeset Symbol DescriptionRefseq Unigene Beta Beta 92809_r_at Fkbp4 FK506 binding protein 4 (59kDa) NM_010219 Mm.12758 0.072658 0.071484 94018_at Ubl3 ubiquitin-like 3NM_011908 Mm.21846 0.038727 0.15764 95465_s_at Cacng6 calcium channel,voltage-dependent, gamma subunit 6 NM_019432 Mm.24750 0.13146 0.04611496608_at Phyh phytanoyl-CoA hydroxylase NM_010726 Mm.27066 0.139810.095547 94478_at Rab5a RAB5A, member RAS oncogene family NM_025887Mm.28872 0.17137 0.046806 94485_at Peci peroxisomal delta3,delta2-enoyl-Coenzyme A isomerase NM_011868 Mm.28883 0.17067 0.02766194489_at Ptp4a1 protein tyrosine phosphatase 4a1 NM_011200 Mm.289090.064335 0.03399 99650_at Csnk1a1 casein kinase 1, alpha 1 none Mm.437370.19561 0.078053 101585_at Pgrmc1 progesterone receptor membranecomponent 1 NM_016783 Mm.9052 0.060712 0.17849 102322_at UgdhUDP-glucose dehydrogenase NM_009466 Mm.10709 0.18791 0.10697 104598_atPtpn16 protein tyrosine phosphatase, non-receptor type 16 NM_013642Mm.2404 0.023338 0.17969 94378_at Rgs16 regulator of G-protein signaling16 none Mm.181709 0.0272 0.12929 97402_at Temt thioetherS-methyltransferase NM_009349 Mm.299 0.19641 0.10905 99064_at Usp4ubiquitin specific protease 4 (proto-oncogene) NM_011678 Mm.3974 0.199060.08681 99076_at Thra thyroid hormone receptor alpha none Mm.265870.060024 0.024133 99959_at Ak4 adenylate kinase 4 NM_009647 Mm.420400.09807 0.015789 100959_at S100a13 S100 calcium-binding protein A13NM_009113 Mm.6523 0.18727 0.11391 102302_at Bckdhb branched chainketoacid dehydrogenase E1, beta polypeptide none Mm.12819 0.142360.16934 103983_at Adh4 alcohol dehydrogenase 4 (class II), pipolypeptide NM_011996 Mm.25845 0.19113 0.032683 92532_at Avpr1a argininevasopressin receptor 1A NM_016847 Mm.4351 0.019388 0.071942

To gain insight into the mechanism of circadian transcriptionalregulation at the level of promoter elements, recently availablemammalian genome sequences (Lander et al., Nature 409:860, 2001; Venteret al., Science 291:1304, 2001) were utilized to identify output geneshad response elements in their structural genes indicative of the coreClock/Mop3 complex (CACGTGA; Hogenesch et al., Proc. Natl. Acad. Sci.USA 95:5474, 1998). Probe sets for all cycling genes were mapped toUnigene using BLAST (Altschul et al., J. Mol. Biol. 215:403, 1990) aspreviously described (Hogenesch et al., Cell 106:413, 2001, which isincorporated herein by reference). The first 300 coding nucleotides ofeach complete Unigene cluster that harbored a translational methioninewere used as bait sequences to search the Celera Mouse Assembly (r12masked) using BLAST. Hits were indexed up to 10 kb upstream of thetranslational methionine (when available), and used to find theconsensus Clock/Mop3 consensus site, CACGTGA.

This analysis revealed a circadian distribution of this element in theSCN with a peak at CT10 to CT14. The distribution of occurrences ofClock/Mop3 binding site in upstream regions of SCN cycling genes wasdetermined, and the number of occurrences of the Clock/Mop3 consensussite was counted upstream of the translational methionine, at 2 kb, 3kb, 5 kb, and 10 kb in the structural genes representing transcripts foreach circadian phase. A specificity score was generated that takes intoaccount the length of the promoter sequences retrieved for eachtranscript and the number of cycling transcripts from each cluster. TheSCN cluster harbored a previously identified E-box element in the Per1promoter, as well as several structural genes whose expression wasunknown to be regulated in a circadian fashion or by the Clock/Mop3complex (see Table 6; SEQ ID NOS:1-14; Gekakis, et al., Science280:1564, 1998). These transcripts included a regulator of G-proteinsignaling-16 (Rgs16), presenilin 2 (Psen2), and protein tyrosinephosphatase, non-receptor type 16 (Ptpn16). The presence of this elementin more than one cluster (e.g., CT22) indicates that other cis-actingelements, trans-activating factors, or post-transcriptional mechanismssuch as mRNA stability also are involved in generating coordinatedcircadian output.

TABLE 6 AGCCACGTGAGG Per1 AGCCACGTGAcA Per1 CTTCACGTGAGG Per2CCCCACGTGAAC 1500039N14Rik AGTCACGTGAGC FLJ20093 AAGCACGTGATG Atp6a2AAGCACGTGACT Atp6a2 TAGCACGTGACC Cckar TAACACGTGAGC Ms4a2 CTCCACGTGACAPsen2 TGTCACGTGACT Rgs16 GAACACGTGACT Ub13 AGACACGTGACG Ptpn16GRACACGTGACC M34 element

The expression pattern of SCN cycling genes harboring a Clock/Mop3consensus site was determined, and expression patterns for genesharboring a Clock/Mop3 response element within 3 kb of the translationalmethionine from the CT10 and CT14 phases were identified. Table 6 showsthe sequence and identity of cycling genes harboring Clock/Mop3 site(SEQ ID NOS:1-14). The core CACGTGA Clock/MOP3 consensus site from eachgene in the above cluster is indicated along with flanking nucleotides.Also included is the sequence of two previously described sites from thePer1 promoter and a closely related site from the Per2 promoter. Whentwo sites were found in a single promoter region, both are indicated.Gene names are derived from Refseq (Pruitt et al., Trends Genet. 16:44,2000).

Protein Biosynthesis in the SCN

To investigate the physiology under circadian control in the SCN, thecycling genes were organized by circadian phase. This analysis revealedthat the largest group of coordinately cycling transcripts share acommon function in protein synthesis. More than 20 transcriptsrepresenting cytoplasmic ribosomal protein components and 13 transcriptsrepresenting mitochondrial ribosomal proteins showed coordinate cyclingwith a peak phase of expression at CT22 (see Panda et al., supra, 2002b;see FIG. 3A, showing that ribosomal protein transcripts and Sui1, andFIG. 5C, showing that protein processing components (NAC, Srp14, Srp9,Sec61γ) peak during night in the SCN, but not in liver (compare FIGS. 3Band 3D, respectively). The first and rate-limiting step in ribosomebiogenesis in nucleoli is the synthesis of ribosomal RNAs mediated bythe multisubunit RNA polymerase I and its accessory factors (Larson etal., Biochem. Cell Biol. 69:5, 1991). While none of the core RNA Pol Isubunits exhibited any significant transcriptional rhythm, a componentshared by all three RNA polymerases-metallothionein 1activator-exhibited a rhythm in peak phase with the ribosomal proteintranscripts. Transcriptional control of a common subunit of all threepolymerases can ensure coordinated regulation of both rRNA and ribosomalprotein transcription. Additionally, transcript levels of TAF Ib, whichis a component of the SL1 complex that recruits RNA Pol I to rRNApromoters, and topoisomerase I, which is essential for RNA pol Imediated transcription, cycle in similar phases.

The control points in ribosome biogenesis and the half-lives ofribosomes exhibit great variability with tissue types. As such, SCNneurons may have adapted a temporal component of ribosome turnover topossibly enhance protein synthesis. In support of this role, diurnalchanges in morphology and size of nucleoli in the rat supraoptic nucleusand superior cervical neurons have been observed (Seite and Pebusque,Chronobiol. Internatl. 2:69, 1985; Bessone and Seite, Cell Tiss. Res.240:393, 1985). Proper initiation complex formation and fidelity oftranslational initiation is another mechanism adapted by organisms toenhance protein synthesis, particularly during stress. One key regulatorof this mechanism is Sui-1 (Cui et al., Mol. Cell. Biol. 18:1506, 1998),which was first identified in yeast. Transcription of Sui-1 is clockregulated in Arabidopsis (see Harmer et al., supra, 2000) and, asdisclosed herein, in Drosophila (Example 1) and in mouse, including inboth liver and SCN. In the SCN, however, the peak phase of Sui-1expression is delayed 8 hr from the peak phase of ribosomal proteins. Assuch, cycling of both ribosome biogenesis and of Sui-1 can be involvedin enhancing translation at different times of day. Importantly, troughlevels of several of these key components remained relatively high,indicating that a basal level of protein synthesis is sustainedthroughout the circadian cycle. Finally, none of these ribosomalcomponents exhibited any significant circadian transcriptional controlin liver, indicating that temporal regulation of protein synthesis inthe liver offers no advantage (see Panda et al. supra, 2002b, FIG. 3B).The results of in situ hybridization, which was performed as previouslydescribed by Bunger et al. (Cell 103:1009, 2000, which is incorporatedherein by reference) revealed a signal from the SCN region at peak andtrough levels of expression supports cycling of the ribosomal proteingene Rp141 (see Panda et al., supra, 2002b; FIG. 3F).

Several genes involved in steps subsequent to translation are also clockregulated, including gene products that participate in protein folding,targeting to endoplasmic reticulum (ER), post-translation modification,and vesicle transport. Shortly after initiation of translation, twodifferent cytoplasmic complexes, dimeric nascent polypeptide-associatedcomplex (NAC), and multimeric ribonucleoprotein complex signalrecognition particle (SRP), compete for binding to the nascentpolypeptide exiting the ribosome (Raden and Gilmore, Mol. Biol. Cell9:117, 1998). SRP selects signal-containing ribosomes for targeting,while binding of NAC prevents targeting of signal peptide-less nascentchains to the ER membrane. Once SRP binds to and docks proper ribosomesto the ER, the subsequent step of protein translocation requires thetrimeric Sec61p complex. Oligomers of the Sec61 complex form atransmembrane channel where proteins are translocated across andintegrated into the ER membrane (Jungnickel and Rapoport, Cell 82:261,1995). The Sec61 complex has also been implicated in translocation ofmisfolded proteins from the ER to the cytosolic protein degradingmachinery, thereby ensuring folding of newly synthesized proteins withfidelity (Romisch, J. Cell. Sci. 112:4185, 1999). As disclosed herein,constituents of NAC, SRP (SRP9, SRP14) and Sec61γ exhibited coordinatedcycling in phase with the ribosomal cluster. In this manner, proteinsynthesis and processing are circadian regulated in the SCN to ensurethat this process is coordinately regulated throughout the night.

Redox and Energy Flux in the SCN

The observation that several genes involved in energy production exhibitcircadian rhythms in their steady state mRNA levels indicates that thisprocess is under clock control. These include components involved incarbon utilization, oxidative phosphorylation, and interconversion ofnucleotide triphosphates. The transcripts of three major enzymes,hexokinase1 (Hk1), malate dehydrogenase, and mitochondrial3-ketoacyl-CoA thiolase cycled in the SCN. This results is consistentwith previous observations that 2-deoxyglucose utilization exhibits amarked circadian oscillation (Schwartz and Gainer, Science 197:1089,1977). While Hk1 and malate dehydrogenase mediate the use of glucose asan energy source, mitochondrial 3-ketoacyl-CoA thiolase regulates theuse of ketone bodies as the major energy source in neurons. Reducingpower derived from the breakdown of glucose and ketone bodies isultimately used in mitochondrial oxidative phosphorylation to generateATP. An example of a gene involved in interconversion of NTPs isnucleotide diphosphate kinase 3, ndk3, which, as disclosed herein, iscircadian regulated and peaks at CT22. In addition to these genes, morethan 20 components of mitochondrial energy production were coordinatelyregulated and clustered at CT22 (see Panda et al., supra, 2002b; FIGS.5A and 5B). FIG. 5A illustrates the mitochondrial oxidativephosphorylation pathway, i.e., transfer of electron from NADH tomolecular oxygen, with concomitant ATP production, which occurs in themitochondrial membrane in four multi-subunit complexes. Components ofthese complexes are encoded by both the mitochondrial and nucleargenomes. FIG. 5B shows that at least seven nuclear components of complexI, one each of complex II and complex III, and six of complex IV peakduring night. The resulting proton motif force is used by multi-subunitF-type ATPases to generate ATP from ADP. Three components of the F-typeATPase, and mitochondrial ADP/ATP translocase 2 peak during night. Datatraces are in blue, genes involved in carbon substrate metabolism are inorange. An in situ hybridization signal of 2900010C23Rik was observed. Aputative NADH dehydrogenase (ubiquinone) flavoprotein 2 (Ndufv2)supports its cycling. These results indicate that expression of thesegenes anticipate impending diurnal neuronal activity.

Cell Signaling

An important circadian output cluster in the SCN includes genes involvedin neurosecretory processes and signaling. Coordinated expression oftranscripts from genes involved in prohormone processing, vesicletransport and fusion, and late endosomal processing, occurred during theanticipation of dawn and dusk (see Panda et al., supra, 2002b; FIGS. 4Ato 4C). In situ hybridization signals obtained for Sgne1 (GenBank Acc.No. NM_(—)009162), secretogranin III (Sg3; NM_(—)009130), and Kcnma1(NM_(—)010610) supported their cycling. A secretory granule protein,Sgne1, previously shown to activate prohormone convertase 2 (PC-2; Braksand Martens, Cell 78:263, 1994), exhibited a circadian rhythm in itstranscript expression. PC-2 has been implicated in the processing ofseveral neuropeptides including somatostatin (Mackin et al.,Endocrinology 129:2263, 1991), an inhibitory neurotransmitter that alsois circadian regulated to the same phase as Sgne1. As such, Sgne1 can beinvolved in accentuating the circadian expression of somatostatin,resulting in a protein rhythm with a peak at CT4 (see Shinohara et al.,Neurosci. Lett. 129:59, 1991). Another secretory granule protein,secretogranin III (Scg3), also exhibited clock regulation.

Several components involved in vesicle trafficking were identified ascircadian regulated to two distinct phases. AP4-sigma, which is involvedin trans-Golgi cycling (Hirst et al., Mol. Biol. Cell 10:2787, 1999),synapsin 1, which is implicated in maintenance of a release ready poolof presynaptic vesicles (Li et al., Proc. Natl. Acad. Sci. USA 92:9235,1995), and Vps29, which is involved in recycling components from theendosome to the trans-Golgi (Seaman et al., J. Cell Biol. 137:7992,1997), all cycled with the same phase as Sgne1 and somatostatin. Incontrast, Snap25 and Munc18c (Jahn, Neuron 27:201, 2000), which mediatevesicle fusion, Eps15, which is implicated in vesicle recycling (Salciniet al., Nat. Cell Biol. 3:755, 2001), and Vps4b, which is involved inlate endosomal vesicle transport (Yoshimori et al., Mol. Biol. Cell11:747, 2000), all cycled with a peak in the late subjective day, CT10.Finally, the mRNA level of several prohormones and peptideneurotransmitters such as proopiomelanocortin (Pomc), adenylate cyclaseactivating polypeptide 1 (Pacap), cholecystokinin, somatostatin, andPDGF-a exhibited clock regulation in the SCN.

Additional molecules, such as arginine-vasopressin (AVP), vasopressinreceptor-1A, somatostatin, PACAP-1, enkephalin, galanin, calcitonin-generelated peptide (CGRP), were reported to cycle at the mRNA levels in SCNtissue (Ibata et al., Front. Neuroendocrinol. 20:241, 1999). In additionto confirming many of these genes in the dataset obtained in this study,rhythms in mRNA levels of genes involved in biosynthesis of non-peptideneurotransmitters such as GABA, histamine, and dopamine also wereobserved. These results indicate that clock regulation ofneurotransmission is more elaborate than previously thought, and alsounderscore the importance of neurosecretion as an important function ofSCN physiology and draw a direct connection to the circadiantranscriptional apparatus.

Circadian Gene Regulation in the Liver

As the master regulator of circadian rhythms in mammals, the SCN mustcoordinate peripheral physiologies under its control. Behavioral controlof feeding, for example, must coordinate with metabolic processes toachieve efficiency in nutrient uptake and utilization. This regulationneed not be direct, as the SCN can regulate liver circadian function byregulating feeding behavior. The SCN pacemaker communicates with thearcuate nucleus (ACN), which is the hypothalamic appetite controlcenter. Rhythms in various orexigenic (NPY, AGRP, Galanin, etc.), andanorexogenic (leptin, insulin, IMSH, glucagon, etc.) signals and plasmahormonal signals ultimately result in a feeding rhythm that is entrainedto activity-arousal mechanisms in the light-dark cycle (Schwartz et al.,Nature 404:661, 2000). Rodents entrained to a light-dark cycle andsubsequently free run in constant darkness exhibit a rhythm in theirfeeding, where they consume more than 70% of their daily food intakeduring nighttime activity (Poirel and Larouche, Chronobiologia 13:345,1986). Food intake may in turn entrain the peripheral clock in theliver. Recent evidence supports this hypothesis by showing that theliver clock can be dissociated from the central oscillator in SCN byrestriction of feeding (Stokkan et al., Science 291:490, 2001).

Energy Metabolism

Circadian rhythms were detected in the transcription of many genesinvolved in nutrient uptake and utilization. Temporal regulationoptimizes use of absorbed nutrients for energy generation and storageduring feeding, and a switch to stored and alternative energy sourcesduring fasting. Coordinated circadian regulation of glucosetransporters, glucagon receptor, and rate-limiting steps in themetabolism of hexose sugars were observed with a peak phase ofexpression in early evening (see Panda et al., supra, 2002b; FIG. 6A,which provides an overview of glucose and fatty acid metabolism in liver(cycling enzymes and transporters are shown in blue), and FIG. 6B, whichdemonstrates that components of gluconeogenesis, glycolysis, and fattyacid metabolism peak during night, when the animals consume the majorityof their diet; GLUT1 and GLUT5 are known to cycle in intestine duringnight (Rhoads et al., J. Biol. Chem. 273:9510, 1998; Castello et al.,Biochem. J. 309:271, 1995); see, also, FIG. 6C, which demonstrates thata putative regulator of fatty acid utilization, lipin (Peterfy et al.,Nat. Genet. 27:121, 2001), cycles with a peak expression during day;cycling of lipin from microarray dataset (in purple, and 2nd y-axis) wasconfirmed independent by real time PCR (in black; Bunger et al., supra,2000)).

This regulation facilitates glycogen synthesis during feeding and use ofhexose sugars as a primary energy source for up to few hours in thepost-adsorptive period (Arias, et al., “The Liver: Biology andPathophysiology” 4th ed. (Lippincott Williams &Wilkins, Philadelphia,Pa., 2001). Similarly, the clock facilitates the use of short- andmedium-chain fatty acids during nighttime feeding and of stored verylong-chain fatty acids during the day.

Cholesterol Metabolism

Transcription of genes involved in cholesterol biosynthesis andmetabolism also was under circadian control. In the liver, a significantportion of acetoacetyl CoA is converted to3-hydroxy-3-methylglutaryl-CoA (HMGCoA), the starting compound incholesterol biosynthesis. Its metabolism is subject to clock regulationacross species, as in plants (Harmer et al., supra, 2000), flies(Example 1), and mice, the transcription of HMGCoA lyase is under clockcontrol. In mammals, the conversion of HMGCoA to mevalonate by HMGCoAreductase (HMGCoAR) is an additional clock-controlled step (Shapiro andRodwell, Biochem. Biophys. Res. Comm. 37:867, 1969). Clock control ofthis step has long been thought to underlie the circadian rhythm inplasma cholesterol level in mammals. However, four additional enzymes ofcholesterol synthesis also exhibited coordinated cycling of theirtranscript levels (see Panda et al., supra, 2002b; FIG. 7A, whichprovides an overview of cholesterol synthesis pathway in animals(Michal, “Biochemical Pathways”, G. Michal, Ed. (John Wiley & Sons,Inc., New York, 1999)); reactions from 3-carbon acetyl CoA up to15-carbon (C15) trans-trans farnesyl-PP take place in peroxisome andcytosol; steps after the clock mediated condensation of two C15 units toform squalene proceed on the ER; degradation of cholesterol producesdifferent steroids, and finally bile acid; and FIG. 7B, whichdemonstrates that enzymes of cholesterol biosynthesis (in blue) peakduring the night). Coordinated cycling of several biosynthetic enzymescan ensure a tight clock regulation of cholesterol production. Whilecholesterol synthesis is phased to the subjective day when dietarycholesterol supply is low, the degradation products of cholesterol(steroids and biles) are produced at different times of the day.Reflective of this, transcripts of many cytochrome P450 gene productsand enzymes involved in cholesterol catabolism were found to be clockregulated and phased to different times of the day (see Panda et al.,supra, 2002b; FIG. 7A, and FIG. 7C, which demonstrates that enzymes ofcholesterol degradation peak at different times of the day; see, also,Kornmann et al., Nucleic Acids Res. 29:E51, 2001); see, also, FIG. 7D,demonstrating that transcription of three membrane channel proteins,Mrp2, Slc22a1, and Slc22a2, which transport bile acids and organiccations, cycle; and FIG. 7E, showing a cycling trace of Cyp7a (in blue)and was confirmed by real time PCR (black).

Bile Acid and Xenobiotic Metabolism

Transcripts from genes involved in several aspects of bile acidmetabolism also were under circadian control. The liver is the onlyorgan that converts cholesterol to bile acids and uses a set of enzymeswith broad substrate specificity for the breakdown and excretion ofcholesterol and of many xenobiotics. This process has been described astwo phased, with the first phase consisting of side group oxidation andhydroxylation, and the second involving the addition of a polyatomicgroup such as sulfate, glutathione, glucuronate, or an amino acid suchas glycine or taurine. These processes change the bioactive propertiesof many substrates and enhance their solubility at acidic pH. More than10 cytochrome P450 genes and related genes mediating the phase Ioxidation of cholesterol were found to be clock regulated. Synthesis ofconjugation partners such as taurine and glycine or enzymes of thesecond phase biotransformation, such as GST also were under clockcontrol. Cysteine dioxygenase (Cdo) catalyzes reduction of cysteine to3-sulfinoalanine, whose subsequent metabolism produces taurine andsulfite (Michal, supra, 1999).

Enzymes mediating the activation of sulfite also were clock regulated,as the enzyme 3′-phosphoadenosine 5′-phosphosulfate synthase 2 (Papss2),which mediates this conversion of sulfite to high energy PAPS, exhibitedclock regulated transcription. Methylation of many xenobiotics alsoalters their activity, and, in this respect, four methyltransferases,including betaine-homocysteine methyl transferase (Bhmt), nicotinamideN-methyltransferase (Nnmt), thioether S-methyltransferase (Temt), andthiopurine methyltransferase (Tpmt) exhibited circadian regulation.

The activities of most methyltransferases are fine-tuned by cellularconcentrations of S-adenosylhomocysteine (SAH). Clock regulatedtranscript levels of SAH hydrolase, which catalyzes the reversiblehydrolysis of SAH to adenosine (Ado) and L-homocysteine (Hcy), also wasclock regulated. This reaction regulates the intracellular SAHconcentration.

Conjugated bile acids and biotransformed xenobiotics are excreted fromhepatocytes by different membrane transporters, some of which areregulated by the circadian clock. The organic cation transportersSlc22a1 and Slc22a2, which transport choline and polyamines, alsoexhibited clock regulation at transcript level. Substrates for Mrp2include bile acid conjugates, glutathione-, glucuronate-, and anionicconjugates of both endobiotics and xenobiotics (Kullak-Ublick et al., J.Hepatol. 32:3, 2000). Clock regulated transcription of components ofxenobiotic metabolism and excretion can account for observedchronotoxicity of a large number of drugs and drug metabolites (Focan,Pharmacol. Ther. 67:1, 1995).

Intermediate Metabolism

In addition to the well described clock regulation of steroidmetabolism, by the clock, (Layery et al., Mol. Cell. Biol. 19:6488,1999; Wuarin et al., J. Cell Sci. Suppl 16:123, 1992), the present studyrevealed cycling transcript levels of many enzymes involved inintermediate metabolism. The liver is the major site of synthesis ofmany bioactive molecules such as nuclear receptor ligands (e.g.,tri-iodo-thyronine, “T3”), retinoid, polyamines, cofactors, and theoxygen carrier heme. Retinoic acid and T3 can bind directly to nuclearreceptors and affect transcription. The thyroid gland releases inactivethyroxine (T4) into blood circulation, which is deionized to active T3by the liver enzyme deiodinase-1 (Dio1). Clock regulated synthesis andrelease of T4 from the thyroid has long been considered as theunderlying mechanism in maintaining a daily rhythm in serum T3. Thisproposed mechanism, however, fails to explain a daily rhythm of plasmaT3 in hypothyroidism patients receiving exogenous T4. Theclock-regulated hepatic transcript levels of Dio1 identified in thedataset obtained in the present study better explains the abovedescribed clinical observation.

Transcript levels of two enzymes, retinol dehydrogenase 7 (Rdh7), andretinal short-chain dehydrogenase/reductase-1 (Rsdr1-pending), alsocycled. Clock regulation of these two enzymes, which act upon all-transretinal produced in a one step reaction from dietary ∂-carotene (fromplants), indicates that synthesis of the nuclear receptor ligand,retinoic acid, and of the visual photopigment, retinol, fromall-trans-retinal also is under clock control. In plants, a putative∂-carotene hydroxylase that converts ∂-carotene to zeaxanthine andrelated photoprotective pigments xanthophylls is under clock control(Harmer et al., supra, 2000; Michal, supra, 1999). As such, thecircadian metabolism of ∂-carotene is conserved across organism asdiverse as plants and animals, demonstrating evolutionary pressure inregulated synthesis of major light sensing pigments in such organisms.

In addition to light reactions, ∂-carotene metabolites are also majorscavengers of reactive oxygen species, and help maintain aphysiologically viable redox state. Heme, however, is the mostextensively used biological sensor of cellular redox state. The firstcommitted and physiologically irreversible step in heme biosynthesis isthe condensation of glycine and succinyl coenzyme A to yielddelta-aminolevulinic acid (ALA). Clock control of ALA-synthase 1 (Alas1)transcription in flies and mammals, and of many additional enzymes inthe subsequent reactions in plants, demonstrates that clock-regulatedproduction of the cellular redox sensors is conserved through evolution.

In summary, a global analysis of circadian patterns of transcriptionfrom the core pacemaker in the SCN and from an important physiologicalmediator, the liver, revealed that as much as 10% of the mammaliantranscriptome is under circadian control, that many of these clockcontrolled genes constitute rate-limiting steps in importantphysiological processes, and that the expression of particular subsetsof circadian controlled genes are tissue specific. These resultshighlight the fundamental importance of circadian rhythms to an organismas a whole, and demonstrate that the circadian system has evolved toencompass different signaling pathways and mechanisms to generate tissuespecific transcriptional rhythms. In addition, evolution has devised aneffective circadian control of physiology by targeting key components ofpathways. Thus, just as biochemical pathways have been conserved overmillions of years, circadian control of such pathways also has beenconserved.

The analysis of clock regulated transcription in the fly (Example 1)indicates that several clock regulated pathways, including hemebiosynthesis (Alas1), cholesterol metabolism (HMGCOA lyase),neuropeptide signaling (Dbi), neuronal excitability (kcnmal/slowpoke),energy metabolism (hexokinase), and metabolism (glutathioneS-transferase and cytochrome P450's) has been conserved over more than600 million years of evolution. Thus, cross-species conservation ofclock regulated transcription can suggest a key role of a component in agiven process. For example, the human homologue of a neuronal nicotinicreceptor b2 (Chrnb2), which cycles in mouse SCN, is mutant in nocturnalfrontal lobe epilepsy, suggesting that Chrnb2 is involved in rhythmiccontrol of motor coordination (De Fusco et al., Nat. Genet. 26:275,2000; Phillips et al., Am. J. Hum. Genet. 68:225, 2001). Similarly, theresults in Example 1 indicate that a large conductance calcium activatedpotassium channel (Kcnma1) has a key role in activity rhythms. Conservedclock regulated expression of Kcnma1 in the SCN, and the recent findingof an important role of potassium ions in SCN function indicates thatKcnma1 represents a conserved mechanism for clock coordination andregulation of activity across species.

EXAMPLE 3 Screening Assays for Sleep/Wake Cycle Modulating Agents

This example provides methods for screening BK channels, includingmethods adaptable to high throughput formats.

A small molecule screen for a perturbagen (i.e., an agonist or anantagonists) of a BK channel can utilize a cell line geneticallymodified to express a BK channel by stable transfection or by transienttransfection. Channel activity can be measured, for example, byinvestigating accumulation of a fluorescent indicator dye in the cellsdue to passage through BK channels. These signals can be measured usingfluorescent plate readers such as fluorescence macro-confocal highthroughput screening (FMAT; Applied Biosystems) using a robotic systemthat allows for the assaying of thousands or more of independentperturbagens in an arrayed or other format. By adding one or acombination of small molecules or other perturbagens to wells andscreening them in such format, agonists or antagonists of BK channelscan be identified.

Although the invention has been described with reference to the aboveexamples, it will be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the following claims.

1-38. (canceled)
 39. A method of identifying an agent that can modulatecircadian regulated locomotor activity in a subject, the methodcomprising: contacting a test system comprising a calcium dependentpotassium channel (BK channel) with an agent suspected of having theability to modulate circadian regulated locomotor activity in thesubject; detecting a change in activity of the BK channel in thepresence of the agent as compared to the activity of the BK channel inthe absence of the agent, thereby identifying an agent that modulates BKchannel activity; administering the agent that modulates BK channelactivity to a test subject; and thereafter detecting a change incircadian regulated locomotor activity of the test subject, therebyidentifying an agent that can modulate circadian regulated locomotoractivity in a subject.
 40. The method of claim 39, wherein the BKchannel comprises a Drosophila slowpoke (slo) polypeptide or an orthologthereof.
 41. The method of claim 40, wherein the ortholog is a mammalianortholog of the slo polypeptide.
 42. The method of claim 39, wherein theBK channel comprises a mutant BK channel.
 43. The method of claim 39,wherein the test system comprises a substantially purified BK channelpolypeptide, and wherein said contacting is performed in vitro.
 44. Themethod of claim 39, wherein the test system comprises a membranecontaining the BK channel, and wherein said contacting is performed invitro.
 45. The method of claim 44, wherein the membrane is a muscle cellmembrane or a nerve cell membrane.
 46. The method of claim 39, whereinthe test system comprises a cell delimited by a cell membrane, or a cellmembrane isolated from said cell, and wherein the BK channel isexpressed in the cell membrane.
 47. The method of claim 46, wherein theBK channel is endogenous to the cell.
 48. The method of claim 46,wherein the BK channel is expressed from a heterologous nucleic acidmolecule.
 49. The method of claim 46, wherein the cell is a muscle cellor a nerve cell.
 50. The method of claim 46, wherein the cell is a humancell.
 51. The method of claim 48, wherein the BK channel is a Drosophilaslo polypeptide or an ortholog thereof.
 52. The method of claim 46,wherein the cell further expresses a BK channel binding protein.
 53. Themethod of claim 52, wherein the BK channel binding protein is aDrosophila slob polypeptide or an ortholog thereof.
 54. The method ofclaim 52, wherein the BK channel binding protein is a ∂ subunit of a BKchannel.
 55. The method of claim 39, wherein the agent is apolynucleotide, a peptide, a peptidomimetic, a peptoid, or a smallorganic molecule.
 56. An agent identified by the method of claim 39.57-67. (canceled)